HU219485B - Process for the preparation of 1,4,7,10-tetraaza-cyclodexecane derivatives, their complexes and conjugates with antibodies, and for the preparation of pharmaceutical compositions containing them, and diagnostic compositions comprising complexes or conjugates. - Google Patents

Abstract

The present invention relates to a process for the preparation of compounds of formula (IA), their metal complexes and conjugates of these complexes with antibodies or antibody fragments. In formula (IA), Q is independently hydrogen or - (CHR5) pCO2R, Q1 is hydrogen or -CO2R, wherein R is hydrogen or C1-4 alkyl, provided that Q and Q1 together have at least two and when Q1 is hydrogen, R3 is other than hydrogen, R5 is hydrogen or C1-4alkyl, n is 0 to 5, p is 1 or 2, R2 is hydrogen, nitro, amino or isothiocyanate R3 is 1- C 4 alkoxy or hydroxy, or hydrogen, R 4 is hydrogen, nitro, amino or isothiocyanate, provided that R 2 and R 4 cannot be hydrogen at the same time, but one of R 2 and R 4 must be hydrogen. ŕ

Description

The present invention relates to novel 1,4,7,10-tetraaza-cyclodecane derivatives, their complexes and their conjugates with antibodies.

Functional chelates or bifunctional coordinators are known to be capable of covalently binding to antibodies specific for cancer, cancer cell epitopes, or antigens. Radioactive nucleus complexes of such antibody / chelate conjugates are suitable for diagnostic and / or therapeutic use because they are capable of transporting the radioactive nucleus to a cancer cell [Meares et al., Anal. Biochem. 112: 68-78 (1984); and Krejcarek et al., Biochem and Biophys. Gap. Comm. 77, 581-585 (1977).

Aminocarboxylic acid chelating agents are known and have been the subject of research for many years. Examples are: nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), hydroxyethyl ethylenediamine triacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), trans-1,2-diamino -cyclohexane tetraacetic acid (CDTA) and 1,4,7,10-tetraaza-cyclododecane tetraacetic acid (DOTA). Various bi-functional chelating agents have been described and prepared which are based on aminocarboxylic acid. For example, the cyclic dianhydride of DTPA [Hnatowich et al. Science 220: 613-615 (1983), U.S. Patent 4,479,930] and mixed carboxylic anhydrides of DTPA (U.S. Patent Nos. 4,454,106 and 4,472,509) and Krejcarek et al., Biochem. and Biophys. Gap. Comm., 77, 581-585 (1977)]. When anhydrides are attached to proteins, coupling occurs via an amide bond, leaving four of the original -5-carboxymethyl groups on the diethylene triamine (DETA) backbone (Hnatowich et al., Int. J. Appl. Isot. , 33, 327-332 (1982)]. U.S. Patent Nos. 4,432,907 and 4,352,751 also disclose bifunctional chelating agents useful for attaching metal ions to "organic groups such as organic target molecules or antibodies". As described above, the coupling is also carried out via the amide group of diamine tetraacetic acid dianhydrides. Examples of such anhydrides are dianhydrides of EDTA, CDTA, propylene diamine tetraacetic acid and phenylene-1,2-diamine tetraacetic acid. U.S. Pat. No. 4,647,447 discloses various complex salts obtained from the anion of a complexing acid and used for various diagnostic purposes. It is taught that the coupling takes place via the carboxyl group of the complexing acid, which forms an amide bond.

Paik et al., J. Med. of Radioanalytical Chemistry 57 (12), 553-564 (1980)] discloses the use of p-nitrobenzyl bromide with a "blocked" diethylenetriamine (e.g., bis (2-phthalimidoethyl) amine) followed by deblocking. and carboxymethylation using chloroacetic acid to yield N'-p-nitrobenzyl diethylenetriamine-N, N, N ', N' -tetraacetic acid. In this method, too, since the bonding is via nitrogen, a tetraacetic acid derivative is obtained. Also disclosed are coupling of a bis-functional chelating agent and chelation with indium. Nitrogen substitution is also described (Eckelman et al., J. of Pharm. Sci. 64 (4), 704-706 (1975)], whereby amines such as ethylenediamine or diethylenetriamine are reacted with the appropriate alkyl bromide prior to carboxymethylation. The compound produced is a potent radiopharmaceutical.

Another class of aminocarboxylic acid-based bifunctional chelating agents are known. For example, Sundberg et al., J. Med. of Med. Chem. 17 (12), 1304 (1974)] discloses bifunctional analogues of EDTA. Representative of these compounds are, for example, 1- (p-aminophenyl) ethylenediaminetetraacetic acid and 1- (p-benzene diazonium) ethylenediaminetetraacetic acid. They also disclose the coupling of these compounds to proteins via the para-substituent and the attachment of radioactive metal ions to the chelating group. These compounds are also described in U.S. Patent Nos. 3,994,966 and 4,043,998, and in Biochemical and Biophysical Research Communications 75 (1), 149 (1977). It is important to note that the attachment of the aromatic moiety to EDTA is via the carbon atom of the ethylene diamine backbone. Optically active bifunctional chelating agents of EDTA, HEDTA and DTPA are disclosed in U.S. Patent No. 4,622,420. In these compounds, the aromatic group (which contains the functional group required for protein coupling) is linked by an alkylene group to the carbon atom of the polyamine, which contains the chelating group. Such compounds are also disclosed in U.S. Patent No. 4,647,447, International Patent Application WO 86/06384, and Brechbiel et al., Inorg. Chem. 25: 2772-2781 (1986).

Recently, certain macrocyclic bifunctional chelating agents and their copper chelate conjugates have been disclosed for diagnostic and therapeutic purposes in U.S. Patent No. 4,678,667 and to Moi et al., Inorg. Chem., 26, 3458-3463 (1987). The aminocarboxylic acid group is coupled to the bifunctional chelating molecule via the ring carbon atom of the cyclic polyamine backbone. Thus, the linker moiety is attached at one end to the ring carbon atom of the cyclic polyamine and at the other end to a functional group capable of reacting with the protein.

Another notable group of bifunctional chelating agents are compounds in which the chelating moiety, i.e., the aminocarboxylic acid, is attached via a nitrogen atom to the functional group of the molecule, which molecule also has the ability to react with the protein. For example, Mikola et al. (WO 84/03698) disclose a bifunctional chelating agent which has been reacted with p-nitrobenzyl bromide and DETA2.

EN 219,485 Β followed by treatment of the resulting compound with bromoacetic acid and its conversion into the aminocarboxylic acid. The nitro group is reduced to the corresponding amine group and then converted to the isothiocyanate group by reaction with thiophosgene. These compounds are bifunctional chelating agents capable of chelating with lanthanides, which can then be conjugated with diororganic molecules and used as diagnostic agents. After the linker moiety of the molecule is attached via one of the nitrogen atoms of the aminocarboxylic acid, a potential aminocarboxylic acid group is lost for chelation. In this way, DETA-based bifunctional chelates are obtained which contain four (not five) acid groups. In this way, this group of bifunctional chelating agents is similar to those in which binding to the proteins occurs via the amide group and so also a carboxyl chelating group is lost.

A recent publication by Caney, Rogers and Johnson [3rd. International Conference on Monoclonal Antibodies Fór Cancer; San Diego, Califomia 2 / 4-6 / 88, titled "Absence of Inrinsically Higher Tissue Uptake I Write Indium-111 Labeled Antibodies: Co-Administration of Indium-111 and Iodine-125 Labeled B72.3 in a Nude Mouse Model" and "Influence of Chelator Denticity on the Biodistribution of Indium-111 Labeled B72.3 Immunoconjugates in Nude Mice", which describes the biodistribution of indium-111 complexed with EDTA and DTPA bifunctional chelating agents. The aromatic ring is attached to the EDTA / DTPA moiety via an acetate methylene group. DK Johnson et al. Also recently described EDTA and DTPA bifunctional derivatives (Florida Conf. On Chem. In Biotechnology, April 26-29, 1988, Palm Coast, FL), where the p-isothiocyanato-benzyl moiety is one of the carboxymethyls. is attached via a methylene carbon atom. Hunt et al., U.S. Patent Nos. 4,088,747 and 4,091,088, previously disclosed ethylene diamine diacetic acid (EDDA) -based chelating agents wherein the aromatic ring to the EDDA moiety is an alkylene or an acetate methylene moiety. connected through a group. These compounds are useful as dormants for studying hepatobiliary functions. The preferred metal is technetium-99m. Indium-111 and indium-113m are also preferred radioactive nuclei for imaging.

It is an object of the present invention to provide complexes which do not dissociate easily, which are capable of rapidly excreting the entire body except for the desired tissues, and to provide conjugates of these complexes with antibodies which produce the desired effect.

The figures of the present invention relate to:

1-7. 15-21. Figures shows the biodistribution ^{of 153} Sm, had been added as a conjugate of the invention containing ¹⁵³ Sm. CC ₄₉ -IgG antibody was used in the conjugate. Biodistribution was determined in nude mice bearing LS 174-T tumor.

8-14. 22-28. Figures ¹⁵³ Sm biodistribution is shown which has been added in the form of a conjugate of the invention containing ¹⁵³ Sm. The conjugate contains the antibody fragment CC ₄₉ -F (ab ') ₂ . Biodistribution was determined in nude mice bearing LS 174-T tumor.

29-34. Figures are shown in biodistribution of ¹⁷⁷ Lu ¹⁷⁷ Lu (PA-DOTMA) - or ¹⁷⁷ Lu (PA-DOTA) -containing conjugates. The conjugates used CC ₄₉ -IgG antibodies. Biodistribution was determined on LS 1174-T tumor bearing (balb / C) mice.

Surprisingly, it has been found that the complexes and / or conjugates of the invention are relatively stable (i.e., not easily dissociated) and some are excreted very rapidly from the body and from certain non-target organs such as the liver, kidney and bone.

It is within the scope of the present invention to provide novel bifunctional chelates, each of which contains a chelating function and a chemically reactive moiety that is capable of covalently linking it to a biomolecule. It is also within the scope of the present invention to provide various bifunctional coordinator (BFC) metal complexes and coupling these complexes to antibodies to obtain radiolabeled antibodies and / or fragments such as samarium-153, lutetium-177 and yttrium-90. for diagnostic and / or therapeutic purposes.

The present invention relates to novel bifunctional chelating agents which form complexes with metal ions, in particular rare-earth "radioactive" metal ions. Rare earth metal metal ions useful in the present invention include La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y and Se, in particular Sm, Ho, Y, and Lu preferred radioactive rare earth-type metal ions include:., ¹⁵³ Sm, ¹⁶⁶ Ho, *> Y, ¹⁴⁹ Pm, i59Gd, ¹⁴⁰ La, ^L77 Lu, ^L75 Yb, ⁴⁷ Sc, ¹⁴² Pr, in particular, ¹⁵³ Sm, ¹⁶⁶ Ho, 9 ° Y and ¹⁷⁷ Lu. Other radioactive metal ions which may also be used are: ⁴⁷ Sc, ^99m Tc, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁹⁷ Ru, wRh, ^lw Pd, ¹⁹⁷ Pt, «Cu, ¹⁹⁸ Au, ' Au, ⁶⁷ Ga, ⁶⁸ Ga, Ίη, Μη, '^ Sn and ²¹² Pb / ²¹² Bi. The resulting complexes are covalently linked to an antibody or fragment thereof and the conjugates thus obtained can be used for therapeutic or diagnostic purposes. The complexes and / or conjugates can be used either in vivo or in vitro. The conjugates of the invention are preferably used for the treatment of cancers of mammals, especially human subjects. Complexes and / or conjugates of the invention which contain non-radioactive metal may also be used for diagnostic purposes and / or for the treatment of diseases such as cancer. Such use of non-radioactive metals generates hyperthermia with radio frequency (Jpn. Kokai Tokyo Koho JP 61, 158,931) and uses fluorescence-immuno-guided therapy (FIGVS) [K. Petterson et al., Clinical Chemistry 29 (1), 60-64 (1983) and C. Meares et al., Acc. Chem. 17, 202-209 (1984)].

HU 219 485 Β

More particularly, the present invention relates to the preparation of compounds of formula IA wherein Q is independently hydrogen or

- (CHR ⁵ ) _p CO ₂ R,

Q * is hydrogen or -CO ₂ R is in which

R is hydrogen or (C 1 -C 4) -alkyl, provided that Q and Q ¹ together are not at least two hydrogen, and when Q ¹ is hydrogen, R ³ must be other than hydrogen,

R ⁵ is hydrogen or C 1 -C 4 alkyl, n is a number from 0 to 5, p is 1 or 2,

R ² is hydrogen, nitro, amino or isothiocyanate;

R ³ is C 1 -C 4 alkoxy or hydroxy, or hydrogen;

R ⁴ is hydrogen, nitro, amino or isothiocyanate, provided that R ² and R ⁴ cannot be hydrogen at the same time, but one of R ² and R ⁴ must be hydrogen.

Also included within the scope of the invention are pharmaceutically acceptable salts of the compounds of formula (IA) above.

Preferred compounds are those compounds of formula IA wherein R is hydrogen,

^R5 is hydrogen or methyl, n is an integer from 0-5, and

R ² is hydrogen, nitro, amino or isothiocyanate;

R ³ is C 1 -C 4 alkoxy, hydroxy or hydrogen,

R ⁴ is hydrogen, nitro, amino or isothiocyanate, with the proviso that R ² and R ⁴ cannot be hydrogen at the same time, but one of R ² and R ⁴ must be hydrogen.

If desired to prepare a conjugate of the invention can be, other than R ² and R ⁴ nitro groups to be.

The invention further relates to complexes with rare-earth metal-type metals, in particular complexes containing neutral radioactive or charged rare-earth metal-ion, and conjugates of these complexes with antibodies or antibody fragments. The present invention also encompasses compositions comprising these conjugates and pharmaceutically acceptable carriers therefor, particularly preferably liquid carriers. These conjugates and pharmaceutical compositions containing them may be used for diagnostic purposes or for the treatment of diseases, particularly cancerous diseases, comprising administering to mammals an effective amount of the compositions.

Preferred electrophilic groups in the above formulas as R ² or R ⁴ are, for example, an isothiocyanate group, the nucleophilic group being an amino group.

It is also desirable that the type and / or position of the R ² , R ³ and R ⁴ groups be such that they are not appreciably involved in the chelating reaction.

As used herein, the term "mammal" refers to any mammal that feeds on its little milk, preferably warm-blooded mammals, more preferably human. The term antibody includes any polyclonal, monoclonal or chimeric antibody, or hetero-antibody, preferably a monoclonal antibody. The antibody fragment may be a Fab fragment and an F (ab ') ₂ fragment, or any portion of an antibody that has specificity for the desired epitope or epitopes. The metal chelate / antibody conjugate or antibody portion includes the entire antibody and / or antibody fragment, including semi-synthetic or genetically engineered variants thereof.

As used herein, the term "complex" refers to a compound comprising a compound of formula (IA) of the invention containing a rare metal type ion, in particular a radioactive rare metal type metal complex, in which at least one metal atom is complexed. The term radioactive metal ion chelate / antibody conjugate or radioactive metal ion conjugate refers to a radioactive metal ion conjugate that is covalently attached to an antibody or antibody fragment. The term radioactive, when used in combination with a metal ion, refers to isotopes of rare-earth elements capable of emitting particles and / or photons, such as ¹⁵³ Sm, ¹⁶⁶ Ho, ⁹⁰ Y, ¹⁴⁹ Pm, ¹⁵⁹ Gd, ¹⁴⁰ La, ¹⁷⁷ Lu, ib Yb, ⁴⁷ Sc and ¹⁴² Pr. The terms bifunctional coordinator, bifunctional chelating agent, and functionalized chelate are equivalent and refer to compounds containing a moiety capable of binding the metal ion in the form of a chelate. a linker group covalently linked to a chelate moiety which serves to covalently attach the molecule to the antibody or antibody fragment.

Pharmaceutically acceptable salts of the compounds of formula IA are salts that are non-toxic and useful in the therapy or diagnosis of mammals. Such salts are prepared in a known manner, for example by reaction with organic or inorganic compounds such as sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, palmitic acid, palmic acid, muconic acid, , glutamic acid, d-camphoric acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, formic acid, lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, cinnamic acid, or other suitable acid. Pharmaceutically acceptable salts include salts formed with basic compounds, such as salts with any organic or inorganic basic compound. For example, ammonia, alkaline metals, alkaline earth metals may be used.

Or other similar ions. Particularly preferred salts are the potassium, sodium, ammonium or mixtures thereof.

Of course, the compounds of formula (IA) may be in free acid form or in protonated form, e.g. when the carboxylate group is protonated and / or when the nitrogen atom is protonated, e.g. when HCl salts are prepared.

Preferred compounds of formula (LA), which are represented by formula (II), are those of formula (LA)

Q ¹ is hydrogen,

Q is independently hydrogen or

-CHR ⁵ CO ₂ R wherein R is hydrogen or C 1 -C 4 alkyl, provided that at least two of Q are not hydrogen, n is 0-5,

R ² is hydrogen, nitro, amino or isothiocyanate;

R ³ is C 1-4 alkoxy or hydroxy,

R ⁴ is hydrogen, nitro, amino or isothiocyanate;

^R5 is independently hydrogen or

C 1-4 alkyl, with the proviso that R ² and R ⁴ cannot be hydrogen at the same time, but one of R ² and R ⁴ must be hydrogen.

Compounds of formula (II) wherein R ² is hydrogen are replaced by compounds of formula (IV):

Q is hydrogen or -CHR ⁵ CO ₂ R,

R is hydrogen or C 1 -C 4 alkyl, provided that at least two of Q are non-hydrogen atoms, n is an integer from 0 to 5,

R ³ is C 1-4 alkoxy or hydroxy,

R ⁴ is nitro, amino or isothiocyanate; R ⁵ is hydrogen or C 1-4 alkyl.

Further preferred are compounds of formula IA wherein at least one of Q is hydrogen, wherein

Q is hydrogen or -CHR ⁵ CO ₂ R,

Q ¹ is hydrogen or -CO ₂ R,

R is hydrogen or C 1 -C 4 alkyl, provided that at least two of Q and Q ¹ must be different from hydrogen and at least one Q must be hydrogen and Q ¹ and R ^{3 are} not simultaneously may be hydrogen, n is a number from 0 to 5,

R ² is hydrogen, nitro, amino or isothiocyanate;

R ³ is C 1 -C 4 alkoxy, hydroxy, or hydrogen,

R ⁴ is hydrogen, nitro, amino or isothiocyanate;

R ⁵ is hydrogen or C 1 -C 4 alkyl, provided that R ² and R ⁴ cannot be hydrogen at the same time, but one of R ² and R ⁴ must be hydrogen.

Other preferred compounds are those compounds of formula IA wherein Q ¹ is -CO ₂ R, which compounds are represented by formula VI, wherein

Q is independently hydrogen or

-CHR ⁵ CO ₂ R,

R is hydrogen or (C 1 -C 4) -alkyl, provided that at least one of Q is other than hydrogen, n is from 0 to 5,

R ² is hydrogen, nitro, amino or isothiocyanate;

R ³ is C 1-4 alkoxy, hydroxy or hydrogen,

R ⁴ is hydrogen, nitro, amino or isothiocyanate;

R ⁵ is hydrogen or (C 1 -C 4) -alkyl, with the proviso that R ² and R ⁴ cannot be hydrogen at the same time, but one of R ² and R ⁴ must be hydrogen.

Preferred compounds of formula (VI) are those wherein R ³ and R ⁴ are each hydrogen; these compounds are represented by formula (VII) wherein Q is independently hydrogen or

-CHR ⁵ CO ₂ R,

R is hydrogen or (C 1 -C 4) -alkyl, provided that at least one of Q is other than hydrogen, n is an integer from 0 to 5,

R ² is nitro, amino or isothiocyanate;

R ⁵ is hydrogen or C 1-4 alkyl.

Further preferred compounds of formula (VI) are those wherein R ² is hydrogen, these compounds are replaced by formula (VIII) wherein Q is independently hydrogen or

-CHR ⁵ CO ₂ R,

R is hydrogen or (C 1 -C 4) -alkyl, provided that at least one of Q is other than hydrogen, n is an integer from 0 to 5,

R ³ is C 1-4 alkoxy or hydroxy,

R ⁴ is nitro, amino, isothiocyanato,

HU 219 485 Β

R ⁵ is hydrogen or C 1-4 alkyl.

Also pharmaceutically acceptable salts of the above-described compounds of formulas II to VIII are also advantageous.

The bifunctional chelating agents of the general formulas (-) to (VIII) described above are capable of binding chelated metal ions, in particular rare earth metal ions with radioactive properties, which form metal ion chelates (also referred to as complex compounds). These complexes are capable of attaching to reactive carriers such as reactive polymers by the presence of a reactive group, or preferably when R ² and R ⁴ are other than nitro in the complex of formula IA, these compounds are covalently bonded to proteins or, more preferably, to antibodies or can bind to antibody fragments. Accordingly, the compounds of formulas (ΙΑ) - (VIII) are capable of complexing rare-earth metal ions, particularly of the radioactive rare-earth metal ions, and the resulting complexes are covalently bound to antibodies or antibody fragments, which are termed conjugates.

Antibodies or antibody fragments useful in the preparation of such conjugates are prepared in a known manner. For example, high specificity monoclonal antibodies can be prepared by hybridization techniques, such as those described by Kohler and Milstein (Naturre 256: 495-497 (1975); and Eur. J. Immunol. 6: 511-519 (1976)]. The antibodies thus obtained generally have high specificity reactivity. The conjugates containing the radioactive metal ion can be used as antibodies to any desired antigen or hapten. Preferably, monoclonal antibodies or antibody fragments having high specificity for the desired epitope or epitopes are used. For example, antibodies to tumors, bacteria, fungi, viruses, parasites, mycoplasma, differential or other cell membrane antigens, pathogenic surface antigens, toxins, enzymes, allergens, drugs, and any biologically active molecule may be used in the present invention. By way of example, the following antibodies or antibody fragments are mentioned: CC-11, CC-46, CC-49, CC-49 F (ab ') ₂ , CC-83, CC-83 F (ab') ₂ and B72.3. See D. Colcher et al., Cancer Slit. 48, 4597-4603 (August 15, 1988)], antibodies CC-49, CC-83 and B72.3. The B72.3 hybridoma cell line is deposited with the American Type Culture Collection (ATCC) under the number HB 8108. The various CC-labeled antibodies are described in U.S. Patent Application No. 7-073685, filed July 15, 1987, and are available from NTIS. Other rat monoclonal antibodies bind to TAG-72 tumor-associated antigenic epitopes. For a full list of antigens, see, for example, U.S. Patent 4,193,983. The radioactive metal ion conjugates of the present invention are particularly useful in the treatment and diagnosis of various cancers.

Preferred rare-earth-type complexes (lanthanides or pseudolanthanides) of the present invention are novel as follows:

C [Ln (BFC)] (IX) wherein Ln is a rare earth, (lanthanide) ion such as Ce ³⁺ , Pr ³⁺ , Nd ³⁺ , Pm ³⁺ , Sm ³⁺ , Eu ³⁺ Gd + ^3, Tb + ^3, Dy + ^3, Ho ^3+, Er ^3+, Tm ^3+, Yb ³⁺ and Lu ^3+, or pszeudolantanida metal ion, such as Sc ^3+, Y ³⁺ and La ^{3 +} , particularly preferred metal ions are Y ³⁺ , Ho ³⁺ , Lu ³⁺ or Sm ³ +; BFC stands for bifunctional chelate; C is a pharmaceutically acceptable ion or ions that have sufficient charge to neutralize the complex.

When the BFC group contains four or more negatively charged moieties, C represents one of the following cation or cation group: H +, Li ⁺ , Na ⁺ , K +, Rb ⁺ , Cs ⁺ , Mg ²⁺ , Ca ²⁺ , Sr ² +, ba ^2+, NH 4 +, N (CH3) 4+, N (C2H5) 4+, N (C3H7) 4 ^+, N (C4 H _{9) 4} +, As (C ₆ H _{5) 4} +, [(C ₆ H ₅ ) ₃ P =] ₂ N + or other protonated amine groups. If the BFC group contains three negatively charged portions, the presence of C is not required. If the BFC group contains two negatively charged moiety, then C is selected from the anions: F-, Cu, Br, I ~, C1O ₄ ", BF _4, H ₂ PO _4, HCO _3, HCO _2, CH ₃ SO ₃ -, H ₃ CC ₆ H ₄ -SO ₃ , pf ₆ -, ch ₃ co _{2 and} B (C ₆ H ₅ ) ₄ -.

The conjugates and, in some cases, the complexes of the invention are also used in the form of compositions. These formulations contain a compound of Formula IA, an antibody and / or a metal ion, and a physiologically acceptable carrier, excipient. For example, a composition of the invention may comprise a physiologically acceptable carrier, a complex (metal ion + ligand), a conjugate (metal ion + ligand + antibody) or (ligand + antibody). The preparation of such compositions may be carried out in a manner known per se, and may take the form of suspensions, injectable solutions or other suitable forms. The physiologically acceptable suspending medium may be used with or without various additives.

The compositions of the present invention are in solid or liquid form which contain an active radioactive core complexed with a ligand. These compositions may be in kit form, in which the two components (i.e., ligand and metal, complex and antibody, or ligand / antibody and metal) are mixed at the appropriate time before use. The composition also comprises a carrier, either pre-mixed or in kit form, which is generally pharmaceutically acceptable.

Injectable compositions obtained by the process of the invention may be in the form of suspensions or solutions. In the preparation of various formulations, it is generally found that the salt-form compounds are soluble in water6

HU 219 485 Β is higher than that of the acid-form compounds. For solution formulations, the complexes (or separate components) are dissolved in a physiologically acceptable carrier. Such carriers include, for example, a suitable solvent, a preservative such as benzyl alcohol, or a buffer, if necessary. Usually the solvent used is water, aqueous alcohols, glycols, phosphonate or carbonate esters. The aqueous solutions contain no more than 50% by volume of organic solvent.

Injectable suspensions of the present invention generally contain a liquid suspending medium to which various additives may be added. The suspending agent may be, for example, aqueous polyvinylpyrrolidone, inert oils such as vegetable oils or high purity mineral oils, or aqueous carboxymethylcellulose. For example, suitable physiologically acceptable additives are necessary to keep the complexes in suspension. For example, thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin or alginates may be used. Various surfactants may also be used as suspending agents, such as lecithin, alkylphenol, polyethylene oxide adducts, naphthalene sulfonates, alkylbenzene sulfonates or polyethyleneoxy sorbitan esters.

Various materials are used to influence hydrophilicity, density, surface tension, which in some cases facilitate the preparation of injectable suspensions. For this purpose, for example, silicone antifoam, sorbitol or sugars are used.

For therapeutic purposes, an "effective amount" of the compositions is used. The dose required will generally depend on the disease being treated. The compositions are generally used for in vitro diagnosis, but may also be used for in vivo diagnosis. The conjugates or compositions of the invention may also be used for radioimmuno-controlled surgical purposes (RIGS), for example, the following metals may be used: ^99m Tc, ⁱⁿ In, ^n3m In, ⁶⁷ Ga or ⁶⁸ Ga.

The chelates of the invention may also be used for various other purposes such as magnetic resonance imaging, such as Gd + ³ complexes of compounds of formula IA, in particular compounds of formula VI, and in various forms bound to polymeric carriers, e.g. , and by selective extraction to remove lanthanide metal or pseudolanthanide metal ion.

Many of the chelates, complexes, and antibody conjugates of the present invention are more stable and / or have a better biodistribution and / or faster elimination from the body than other similar formulations known hitherto. The invention also relates to processes for the preparation of these chelates, complexes and antibody conjugates.

Preferably, the 12-membered macrocyclic bifunctional chelates of formula (IA) are prepared by monofunctionalizing the free base macrocyclic compound (e.g., 1,4,7,10-tetraazacyclododecane) with the corresponding electrophilic compound, e.g. , appropriately substituted α-halo-carboxylic acid ester). This electrophilic compound must contain a suitable linker moiety or a suitably protected linker moiety that permits covalent attachment of the bifunctional ligand to the protein, antibody or antibody fragment.

It is known in the art that alkylation of mono-N-functionalized polyazacrocyclic compounds results in a mixture of different, hardly separable products [T. Kaden, Top Curr. Chem. 121: 157-75 (1984)]. This was eliminated by the described methods by using a large excess of the macrocyclic compound (5-10 equivalents relative to the electrophilic compound), which favored the formation of the monoalkylated adduct [M. Studer and T. A. Kaden, Helv. Chim. Acta 69: 2081-86 (1986); E. Kimura et al., J. Chem. Soc. Chem. Commun. 1158-59 (1986)].

Other methods include the preparation of mono-N-functionalized polyazacrocyclic compounds using lengthy protective, functionalizing and deprotection reactions [P. S. Pallavicini et al., 1987, J. Amer. Chem. Soc. 109, 5139-44; M. F. Tweedle et al., Eur. Pub. Pat. Appl. No. 0232-751]. Recently, reductive amination of substituted phenylpyruvic acids and amines has been reported in an extract from a recent meeting of the Abbott Laboratory [D. K. Johnson et al., Florida Conf. On Chem. In Biotechnology, April 26-29, 1988, Palm Coast, FI] It is further known that a mono-N-alkylated polyazacrocyclic compound is prepared by that the electrophilic compound is used in an amount of 1 to 5 equivalents relative to the corresponding macrocyclic compound in a solvent that does not promote proton transfer (U.S. Patent No. 289,163, filed December 22, 1988).

In the process of the present invention, the mono-N-substituted poly-macrocyclic compound used as starting material is depicted with a compound of formula (IA '), wherein the substituents have the meaning given by general formula (IA) and this compound is (CHR ⁵ ) _p CO ₂ R.

For the preparation of compounds of formula IA in which R ² or R ⁴ is amino, a compound having R ² or R ⁴ is nitro is reduced, or a compound of formula R 1 in which R is hydrogen is reduced to 1 to 4 carbon atoms. hydrolyzing the compound having an alkyl group (IA), or for the preparation of (IA) wherein R ² or R ⁴ is an isocyanate, R ² or R ⁴ is an amino group is reacted with thiophosgene atom.

I to III. The following Schemes illustrate a general process for the preparation of chelates of the present invention employing the appropriate electrophilic and polyazacrocyclic compounds in various stoichio

EN 219 485 Β at different temperatures and concentrations in a suitable organic solvent. As the organic solvent, any hydrocarbon compound can be used which provides the appropriate solubility, such as acetonitrile, isopropanol, methylene chloride, toluene, chloroform, n-butanol, carbon tetrachloride, tetrahydrofuran, 5% ethanol chloroform, of which: particularly preferred are chloroform, methylene chloride, n-butanol, 1,4-dioxane and acetonitrile. The macrocyclic compounds and the electrophilic compounds are used in stoichiometric or near stoichiometric ratios, so that the corresponding mono-N-alkylated product is swallowed in a single step. The temperature is generally from 0 ° C to the reflux temperature of the solvent, preferably from 0 ° C to 25 ° C. The reaction time is usually 1 to 24 hours.

II. α-haloic acid esters are required to carry out the procedure of Scheme II. A suitable process is the bromination or chlorination of acid halides formed in situ [D. N. Harpp et al., J. Org. Chem. 40, 3420-27 (1975)]. In this method, it is possible to produce only α-halogenated alkanoic acids which even contain a reactive benzyl group. In the general preparation of substituted acid halides, the organic acid is reacted with thionyl chloride or sulfuryl chloride [E. Schwenk et al., J. Amer. Chem. Soc., 70, 3626-27 (1944). In both methods, free carboxylic acid is used as a starting material, which is commercially available.

Polyazamacrocyclic compounds such as 1,4,7,10-tetraaza-cyclododecane may be prepared by known methods (e.g., TJ Atkins et al., J. Amer. Chem. Soc., 1974, 96, 2268-70 (1974)). Richman et al., Org. Synthesis 58, 86-98 (1978)].

The carboxymethylation of the mono-N-functionalized macrocyclic compound can be accomplished by the Desreux method using a bromoacetic acid derivative and a suitable base [J. F. Desreux, Inorg. Chem., 19, 1319-24 (1980)].

Any starting material used in the process of the invention is commercially available or can be prepared according to known methods described in the literature.

Scheme I illustrates the preparation of compounds of formula IA wherein Q ¹ is -CO 2 R, Q is -CH 2 -COOR, and R ³ is hydrogen. Although the reaction scheme is directed to the preparation of a particular compound, other compounds of formula IA wherein Q ¹ is CO ² R and n = 0-5, and the other substituents are as defined above, may be prepared.

II. Scheme _II illustrates the preparation of compounds of formula IA wherein Q is (CHR ⁵ ) _p CO ₂ R wherein R ⁵ is other than hydrogen and R ³ is hydrogen. Although the Scheme illustrates the preparation of a particular compound, other compounds of formula IA can be prepared by the process wherein Q is (CHR ⁵ ) _p CO ₂ R and n = 0-5 and the other substituents are as defined above. .

AII. In Scheme A, the group A '(compound of formula S) in the reagent may be any leaving group preferred for the alpha acids, such as A' being phenyl or CF ₃ .

The use of optically active alkylating agents minimizes the formation of diastereomers and simplifies the process. The separation of the individual diastereoisomers increases the amount of each of the easily purified lanthanide complexes desired for radio-chelation as well as antibody conjugation.

In III. Scheme II illustrates the preparation of compounds of formula IA wherein Q ¹ is CO ² R, R ² is hydrogen, and R ³ is methoxy. Although the Scheme illustrates the preparation of two specific compounds, other compounds can be prepared by the process wherein Q ¹ is hydrogen or (CHR ⁵ ) pCO ₂ R, Q is hydrogen or CO ₂ R, n = 0-5, p = 1 or 2, R ³ is as defined above, R ² and R ⁴ are hydrogen, amino, nitro or isothiocyanate.

The electrophilic compound can also be prepared in a known manner. Such methods are described, for example, in Acc. Chem. 17, 202-209 (1984). The preparation of compounds of formula IA wherein R ² or R ⁴ is an isothiocyanate group is carried out by reacting an amino chelate with R ² or R ⁴ an amino group with thiophosgene (U.S. Patent No. 289,172), December 23, 1988).

The radio package can be obtained in various ways, for example by bombarding the nucleus with neutrons in a nuclear reactor, for example:

Sm-152 + neutron -> Sm-153 + gamma.

Alternatively, the radio packages are obtained by bombarding the nucleus with a linear accelerator or cyclotron. In another method, the radio packages are separated from a mixture of fusion products. The method of preparing the radioactive nuclei is not critical to the compounds of the invention.

The conjugates of the invention may be prepared by first forming the complex and then binding to the antibody or antibody fragment. Thus, in this process, the ligand is first obtained, then complexed with the metal, and then the resulting complex is added to the antibody. Alternatively, in the preparation of labeled antibody conjugates, BFC is first bound to the antibody and then chelated to swallow the radioactive BFC labeled antibody. Any other method by which the conjugates of the invention may be prepared is also within the scope of the invention.

In the following examples, which illustrate the invention in greater detail, the following terms, experimental conditions, are used.

Mass spectral measurements were performed on a Finnigan TSQ mass spectrometer (Q'MS mode) or VG ZAB-MS

HU 219 485 Β high resolution mass spectrometer (fast atom bombardment with xenon, 3: 1 = dithiothreitol: dithiol erythritol).

The Ή and ¹³ C-NMR spectra were recorded on a Varian VXR-300, Bruker APC 300, IBM / Bruker NR-80 or Jeol FX400 spectrometer. All spectra were recorded at 30 ° C unless otherwise noted. The ¹ H-NMR spectrum was recorded at 300 MHz, 80 MHz, or 400 MHz, depending on the device listed above: the ¹³ C-NMR spectrum was also recorded at 75 MHz, 20 MHz or 100 MHz, depending on the device. NMR values reported are δ per TMS (tetramethylsilane) values or, if D ₂ O is used as solvent, per DSS (2,2-dimethyl-2-silapentane-5-sulfonic acid, sodium salt).

Infrared (IR) spectra were recorded on a Nicolet 5SL FT / IR instrument.

Most of the solvents used in the chromatographic methods were Fisher HPLC grade. Ammonium acetate was purchased from Aldrich. The water was purified using the Bamstead NANPOpure ™ water purification system. Preparative chromatography of the organic compounds was carried out either by normal gravity chromatography or by flash chromatography [C. W. Still et al., J. Org. Chem., 43, 2923-24 (1978)]. The following solvent systems were used for the chromatography: Solvent system Components

1. CHCl ₃ : CH ₃ OH: Conc. NH ₄ OH 2: 2: 1

V: V: V

2. CHCl ₃ : CH ₃ OH: Conc. NH ₄ OH 12: 4: 1

V: V: V

3. CHCl ₃ : CH ₃ OH: Conc. NH ₄ OH 16: 4: 1

V: V: V

4. CHCl ₃ : CH ₃ OH: Conc. NH ₄ OH 4: 2: 1

V: V: V

5. CHCl ₃ : CH ₃ OH: Conc. NH ₄ OH 3: 2: 1

V: V: V

6. CHCl ₃ : CH ₃ OH: Conc. NH ₄ OH 7: 3: 1

V: V: V

Saline 7 (0.85% NaCl in distilled water): Conc.

NH ₄ OH

4: 1

V: V

8. CHCl ₃ : CH ₃ OH: Conc. NH ₄ OH 4: 4: 1

V: V: V

In these examples, _Rf values were obtained using these solvent systems and commercially available normal phase silica TLC plates (GHLF 250 micron, Analtech Inc. or Merck Kiesel gel 60F _254). Merck 60.60 Å silica gel was used for preparative column chromatography.

The percentages given in the examples are by weight unless otherwise indicated.

In some cases, the resulting solid was dried in a rotary evaporator (Buchi 461) and / or in a vacuum oven at 55-60 ° C. In some cases, a Virti 10-010 automatic freeze dryer or a Speed Vác ™ concentrator was used to remove the solvent.

Samarium-153 and lutetium-177 were obtained from Research Reactor, University of Missouri (Columbia, MO), and Yttrium-90 was obtained from Oak Ridge National Laboratory.

1- (4-Isothiocyanato-benzyl) -diethylene-triamine-pentaacetic acid (SCN-Bz-DTPA) is described by M. W. Brechbiel et al., Inorg. Chem. 25, 2772-2781 (1986)] and the product was purified by anion exchange HPLC (Q-Sepharose ™) to give a single product. The reagents used are listed below, with the places of purchase in parentheses: Ν-2-hydroxyethylpiperazineaz-2-ethanesulfonic acid (HEPES), free acid or sodium salt (Bering Diagnostics, La Jolla, CA), sodium citrate (certified by Fisher Scientifc), thiophosgene (Aldrich Chemicals), ammonium acetate, sodium acetate, ethylenediaminetetraacetic acid, phosphate buffer (Sigma Diagnostics).

The HPLC column used was Handpacked Q-Sepharose ™ (Pharmacia) at either 1.5 cm x 25 cm or 2.5 cm x 25 cm; Zorbax ™ BIO Series GF-250 (9.4mm x 25cm) from Du Pont Instruments; Vydac ™ (trade name of Separation Group, Hesperia, CA) protein C-4 (4.6 mm x 25 cm) manufactured by Separation Group (Hesperia, CA); and Mono-Q ™ by SP-Sephadex ™ (a trademark of Pharmacia Biotechnology Products) manufactured by Pharmacia. Sep-Pak ™ (trademark of Waters Associates) C-18 Stuff, available from Waters Associates (Milford, MA), Sephadex ™ G-25 Disposable Column (2.2 mL) from Isolab Inc. (Akron, OH) and Centricon ™ -30 (Amicon Division, WR Grace & Co., Danvers, MA) Microconcentrator, available from Amicon.

Five different HPLC systems were used for analysis and sample separation:

System I: LKB 2150 Pump, 2152 Controller, UV Detector - LKB 2238 W Cord, Barthold LB 506 A HPLC Radioactivity Monitor (of the International Berthold Group) and Gilson Fraction Collector 201-202 (Gilson International, Middleton, WI) ;

System II: automatic sample dispenser, WISP 710 B, two pumps (model 510) and automatic gradient regulator (Waters Associates), Perkin-Elmer LC-75 spectrophotometric detector, Beckman model 170 radioisotope detector and fraction collector (Frac-100, Pharmacia);

System III: two Waters M-6000A pumps, a Waters 660 solvent programmer, a Waters Model 481 Variable Wavelength Detector, a Waters Data Module and an ISCO fraction collector;

HU 219 485 Β

System IV: DionexBioLC ™ system equipped with a variable wavelength UV detector; and

System V: Waters Model 590 Pump, ISCO Model 2360 Gradient Programmer and Dionex Variable Wavelength Detector.

A Sorvall RT 6000B (refrigerated centrifuge, manufactured by Du Pont) was used for centrifugation and concentration. Volatile solvents were removed using a Speed Vac concentrator (Savant Instruments Inc., Hicksville, Y.). Radiological measurements were performed with a Capintec ™ radioisotope calibrator CRC-12 or liquid scintillation (Beckman LS 9800, Beckman Instruments, Inc., Irvine, CA) or a Canberra 35+ multi-channel analyzer, 3 in. thallium-containing sodium iodide hollow crystal inner partition.

In the examples for the preparation of complexes, the percentage of the complex was determined by the following known ion exchange method.

A 10 mL plastic column was filled with 1-2 mL of SP-Sephadex ™ C-25, previously swollen in water, and excess water was removed by applying pressure to the top of the column. 15 μΐ of the test solution was added to the resin at the top of the column and 2 ml of a 4: 1 (v / v) isotonic saline: concentrated ammonium hydroxide eluant was added. The eluent was collected in a graduated tube. A further 2 ml of eluent was added. After collecting the eluent in a second tube, the resin was dried by applying pressure to the top of the column. The dried resin was then transferred to a third measuring tube. The activity of the measuring tubes was determined with a NaI counter connected to a Canberra multi-channel analyzer. The amount of metal bound in the complex was expressed as a percentage of the total activity determined in the eluent. The amount of uncomplexed free metal is expressed as a percentage of the total amount of metal remaining on the resin.

Removal of uncomplexed metal by ion exchange

A 10 mL plastic column was filled with 0.5 mL SP-Sephadex ™ C-25 resin in water. Excess water was removed by centrifugation at 3000 rpm for 4 minutes. 200-500 μΐ of the complex compound solution was then added to the top of the resin and centrifuged again at 3000 rpm for 4 min. The uncomplexed metal remaining on the column as well as the complexed metal was removed by elution with solvent.

Preparation of yttrium complex

A complex compound was prepared by preparing a 3x10 ⁴ molar aqueous solution of yttrium [YCl ₃ -6H ₂ O, 303.26 g / mol; or Y (OAc) ₃ , 12.1% H ₂ O]. Radioactive To this solution was added ³ + Y (Oakridge National Laboratories) to provide the desired radioactivity. Thereafter, 10 μΐ of ligand solution (0.03 mol) was added to 990 μΐ of Y ³⁺ solution to provide a 1: 1 molar ratio of ligand: metal. Ten times the ligand solution was used to provide a 10: 1 ligand: metal ratio. The pH was adjusted to 7.4 using microliters of hydrochloric acid or sodium hydroxide. The amount of complexed yttrium in the resulting solution was then determined using the cation exchange method described above.

Production of Samarium Complex

The samarium complex was prepared by the method described above for the preparation of the yttrium complex, except that a 3 χ 10 ^-4 molar solution of samarium was prepared by dissolving 348.7 g / mol Sm ₂ O _{3 in} 0.1 M hydrochloric acid. The radioactive Sm-153 was obtained as a solution of 3 χ 10 ^-4 mol / 0.1 M hydrochloric acid from the University of Missouri Research Reactor, Columbia, Missouri.

Here's what the abbreviations used in the examples mean:

-OAc is an acetate group, -OCOCH ₃ ;

TLC refers to thin layer chromatography;

DI H ₂ O is Nanopure ™ deionized water;

NANOpure ™ water is filtration purified water produced by Barnstead NANOpure ™; ambient temperature is generally 20-25 ° C;

overnight is 9-18 hours;

LC is liquid chromatography;

NBS is N-bromosuccinimide;

MES is 2- (N-morpholino) ethanesulfonic acid;

HPLC refers to high performance liquid chromatography;

PBS is phosphate buffered saline, manufactured by Sigma, 120 mM NaCl, 2.7 mM KCl and 10 mM phosphate buffer, pH 7.4;

SP-Sephadex ™ C-25 Resin means: a cation exchange resin with sulfonic acid functionalities, available from Pharmacia Inc.;

pD is the pH at which hydrogen is deuterated;

DOTA is 1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid;

HEPES represents N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid;

BFC stands for bifunctional chelate;

cyclen is 1,4,7,10-tetraaza-cyclododecane;

PA-DOTA is α- [2- (4-aminophenyl) ethyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid;

PA-DOTMA is α- [2- (4-aminophenyl) ethyl] 1,4.7.10-tetraaza-cyclododecane-1-acetic acid-4,7,10-tris (methyl acetate);

BA-DOTA is α- (4-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid;

OMe-BA-DOTA is α- (5-amino-2-methoxyphenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid;

EA-DO ₃ A is 1- [2- (4-aminophenyl) ethyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid;

DTPA is diethylene triamine pentaacetic acid;

SCN-Bz-DTPA is 1- (4-isothiocyanato-benzyl) -diethylene-triamine-pentaacetic acid;

EDTA is ethylenediamine tetracetic acid; the chelate is the same as the ligand; the complex is the same as the chelate;

Kon a conjugate is a chelate or complex which is covalently linked to an antibody or antibody fragment; and the antibody may be CC-49, CC-83 and B-72.3, as well as ffag fragments thereof such as Fab and F (ab ') ₂ . Other antibodies which may also be used have been mentioned above. The B-72.3 hybridoma cell line is deposited with the American Type Culture Collection under accession number ATCC HB 8108, and the other rat monoclonal antibodies mentioned above bind to TAG-72 tumor-associated antigenic epitopes.

The following examples are by no means limiting but merely illustrative of the invention.

Preparation of starting compounds

Example A Methyl d, l-2-bromo-4- (4-nitrophenyl) butanoic acid methyl tetrachloride (15 ml, 0.2 mole) was mixed with 10.46 ml of nitrogen. g (0.05 mol) of 4- (4-nitrophenyl) butanoic acid.

The resulting solution was heated at reflux for 1 hour, during which time hydrogen chloride and sulfur dioxide gas were evolved. To the warm solution was then added 11 g of N-bromosuccinimide (25 g, 0.06 mol) in carbon tetrachloride (25 ml) and 3 drops of 48% aqueous hydrobromic acid catalyst. A bromine gas evolution is observed. The dark red solution was then refluxed for 35 minutes, cooled, poured into methanol (100 mL) with stirring. TLC analysis (60/40 = ethyl acetate / hexane, v / v) indicated a new product (Rf = 0.69, silica gel plates). Excess solvent was removed in a rotary evaporator and the resulting dark red oil was filtered through silica gel flash chromatography using methylene chloride as eluent. The solvent was then evaporated to leave a clear oil (15.43 g) which was a 45:15 mixture of the title compound and the methyl ester of the starting butanoic acid. The title compound 40 has the following characteristics:

1 H-NMR (CDCl ₃ ):

8.16 (d), 7.38 (d), 4.20 (dd), 3.79 (s), 2.88 (m),

2.38 (m);

³ C-NMR (CDCl ₃ , 75 MHz): 45

169,6,147,5,129,3,123,7, 53,0,44,4, 35,5, 33,0.

Example B Methyl α- [2- (4-Nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1-acetic acid 50

1,4,7,10-Tetraaza-cyclododecane (1.72 g, 10 mmol) and chloroform-stabilized with pentene (17 mL) were stirred with d, 1-2-bromo-4- (4- Nitrophenyl) butanoic acid methyl ester (prepared as in Example A) was added over 5 minutes under nitrogen with stirring. The reaction mixture is then stirred for a further 48 hours at about 25 ° C. TLC analysis (2 solvent systems, Analtech silica gel plates) showed the formation of the title compound (R _f = 0.73). The resulting yellow chloroform solution was then applied to a silica gel column which had been pre-eluted with 5% methanol in chloroform and eluted with 250 ml of 5% methanol in chloroform followed by 2 solvent systems. The pure fractions containing the title compound were combined and concentrated to give 2.15 g (5.46 mmol, 94%) of the title compound as a yellow glass. The chloroform solution of the product was then mixed with diethyl ether to give an analytically pure product as a white powder (m.p. 156-159 ° C).

1 H-NMR (CDCl ₃ ):

8.14 (d), 7.39 (d), 3.71 (s), 3.39 (dd), 2.5-3.0 (m), 2.08 (m), 2.01 ( m);

³ C NMR (CDCl ₃ , 75 MHz):

127.7, 149.3, 146.4, 129.2, 123.6, 62.3, 51.2, 48.9,

47,2,45,8,45,4,3,2,8,30,9

Example C)

2-Bromo-2- (4-nitrophenyl) -acetic acid methyl ester g of p-nitrophenylacetic acid (0.14 mol) and 15.1 ml (0.21 mol) of thionyl chloride in 100 ml of anhydrous in benzene at reflux under nitrogen for 3 hours. The resulting mixture is then evaporated to dryness in vacuo, the residue is dissolved in carbon tetrachloride and stirred under nitrogen at reflux. Bromine (7.2 mL, 0.14 mol) was added in small portions over 3 days under reflux, then the mixture was allowed to cool and methanol (50 mL) was added slowly. The resulting bromo ester (27 g, 71%) was separated by distillation (boiling point = 168 ° C / 1.1 mm).

1 H-NMR (CDCl 3):

8.25 (d), 7.81 (d), 5.51 (s), 3.86 (s).

Example D Methyl α- (4-nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1-acetic acid

529 mg (2.068 mmol) of 2-bromo-2- (4-nitrophenyl) -acetic acid methyl ester (prepared in Example C) are mixed with 20 ml of acetonitrile and added in one portion 354 mg (2.068 mmol) of 1.4,7, 10-tetraaza-cyclododecane in 20 ml of acetonitrile, which still contained enough methanol to form the solution. The resulting pink solution was then stirred for 1.5 hours and then concentrated in vacuo to a small volume at 35 ° C. The resulting crude monoester tetraamine is chromatographed on silica gel (20% w / v NH ₄ OAc / CH ₂ OH). The purified monoester tetraamine is recovered as the triacetate salt which is then converted to the free amine. 270 mg of the material thus obtained are mixed with 3 ml of water at 0 ° C with 10% by weight aqueous potassium carbonate, the pH is adjusted to 11, the resulting alkaline solution is then extracted five times with 10 ml of chloroform, and the chloroform phases are combined. After drying over sodium sulfate and concentration, 160 mg (42%) of the desired monoester tetraamine are obtained.

1 H-NMR (CDCl ₃ ):

8.12 (d), 7.44 (d), 4.75 (s), 3.76 (s), 2.67-2.44 (m);

HU 219 485 Β ¹³ C-NMR (CDCl ₃ ):

171.5, 147.6, 144.0, 130.0, 124.0, 67.0, 49.8, 47.9, 46.9, 46.0.

FAB MS = [M + H] ⁺ = 366.

Example E)

N- (2-Methoxy-5-nitrobenzyl) -1,4,7,10-tetraaza-cyclododecane ml of chloroform was stirred in 2.9 g (16.8 mmol)

1.4.7.10-Tetraaza-cyclododecane, then 2.1 g (8.5 mmol) of 2-methoxy-5-nitrobenzyl bromide dissolved in 20 ml of chloroform are added in one portion. The resulting mixture was then stirred at room temperature for 3 hours, then filtered, concentrated in vacuo and chromatographed (silica gel, three solvent systems) to give a monoalkylated product, m.p. 154-156 ° C, 79% yield.

¹³ C NMR (CDCl 3):

162.47, 140.63, 127.92, 125.49, 124.53, 109.91, 55.88, 53.25, 50.90,47,18,45,45,45,29.

Example F)

N- (2-Hydroxy-5-nitrobenzyl) -1,4,7,10-tetraaza-cyclododecane in 1,4-dioxane (ml) was dissolved in 2.3 g (14 mmol).

1.4.7.10-Tetraaza-cyclododecane, then 1.2 g (7 mmol) of 2-hydroxy-5-nitrobenzyl bromide dissolved in 15 ml of dioxane are added in one portion with constant stirring. After a few minutes, 12 g of potassium carbonate are added and stirring is continued for 1 hour at room temperature. The reaction mixture was then filtered, and the filtrate was concentrated in vacuo to give a semi-solid yellow solid which was chromatographed on silica gel (solvent system 5). The desired monoalkylated product is recovered from the last 1/3 of the yellow eluent, which even contains unreacted amine. This fraction was concentrated and the residual yellow oil was mixed with 100 mL of CHCl ₃ and filtered to remove the CHCl ₃ soluble amine starting material. The final product was then swallowed as a yellow solid (1.2 g, 55%), mp 142-145 ° C.

¹³ C-NMR (D ₂ O);

25.04, 25.28, 26.83, 31.80, 36.54, 101.88, 107.22, 110,92,111,80,115,26,159.99.

Example K)

2-Methoxy-5-nitrobenzylnitrile Sodium cyanide (6 g, 121.9 mmol) was dissolved in water (5.3 mL) and 25 g (101.6 mmol) in ethanol (15 mL) was added in small portions with stirring at 70 ° C. 2-methoxy-5-nitrobenzyl bromide is hot. The reaction mixture was then heated at reflux for 1.5 hours, cooled, filtered, and the filter cake was washed with acetonitrile and the filtrate concentrated in vacuo to give 19.5 g (100%) of 2-methoxy-5-nitrobenzenitrile as a beige color. in the form of a solid.

13C NMR (CDCl3):

161.4, 141.0, 125.7, 124.6, 119.8, 116.5, 110.2,

56,3,18,8.

Example L)

2-Methoxy-5-nitro-phenylacetic acid

19.5 g of the product of the preceding Example G) are mixed with 20 ml of concentrated hydrochloric acid and heated under reflux for 3 hours, after which the crude product is isolated by filtration and washed with water. The solid was then dissolved in hot aqueous sodium hydroxide and filtered hot. The orange solution was cooled, acidified with hydrochloric acid, and the resulting precipitate was filtered off, washed with water and dried to give 15 g (70%) of the title compound as a white solid.

13C NMR (CDC1 ₃ -CD ₃ OD):

127.8, 162.5, 140.7, 126.2, 124.7, 124.1, 109.7,

55.8, 35.0.

Example M d-Bromo (2-methoxy-5-nitrophenyl) acetic acid, methyl ester

2-Methoxy-5-nitrophenylacetic acid (2.266 g, 10.74 mmol) (prepared as in Example H) was mixed with benzene (50 mL) and thionyl chloride (4 mL, 54.8 mmol) was added. A drying tube and a condenser are then placed on the flask and the mixture is refluxed for 2 hours. The resulting yellow solution was concentrated in vacuo, the small residue was dissolved in anhydrous carbon tetrachloride, bromine (0.6 mL, 11.6 mmol) was added and the mixture was refluxed for 2 days at atmospheric moisture. The mixture was then concentrated to a small volume, methanol (50 mL) was added, excess methanol was removed in vacuo and the residue was chromatographed (silica gel, methylene chloride / hexane = 4: 1). The title compound was swallowed as a yellow oil (2.399 g, 74%).

1 H-NMR (CDCl ₃ ):

8.45 (dd), 8.15 (dd), 6.95 (d), 5.75 (s), 3.99 (s), 3.78 (s).

Example N Methyl α- (2-Methoxy-5-nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1-acetic acid

Α-Bromo- (2-methoxy-5-nitrophenyl) acetic acid (2.999 g, 7.89 mmol, prepared as in Example I) was dissolved in chloroform (10 ml) and 2.72 g (15.79 g) in chloroform (50 ml) was added with stirring. mmol)

1,4,7,10-tetraazacyclododecane. The resulting mixture was stirred at room temperature for 2 hours and then the cloudy mixture was concentrated in vacuo to a small volume at room temperature. The crude product was purified by chromatography (silica gel, solvent system 3) to give the title compound as a yellow solid (2.6 g, 83 g).

¹³ C NMR (CDCl ₃ )

170.7, 161.6, 1339.9, 125.0, 1124.5, 124.2, 110.2, 59.5, 55.5, 50.5,48,0,47.3, 45, 6, 44,3,43,5.

Example O d, 1-2-Bromo-4- (4-nitrophenyl) butanoic acid isopropyl ester g (0.1 mol) 4- (4-nitrophenyl) butyric acid dissolved in 10 ml of carbon tetrachloride (30 ml). (4 moles) of thionyl chloride in nit12

EN 219,485 in a hydrogen atmosphere according to the method of Harpp et al. Org. Chem. 40, 3420-27 (1975)]. The resulting solution is then heated at reflux for 1 hour, at which time hydrogen chloride gas and sulfur dioxide gas are suddenly released. N-bromosuccinimide (20 g, 0.12 mol) dissolved in carbon tetrachloride (50 ml) was added dropwise and 8 drops of 48% aqueous hydrobromic acid catalyst were added dropwise before the bromine was released. The dark red solution was then heated for a further 35 minutes and then cooled and poured into 400 ml of isopropanol with stirring. TLC analysis (methylene chloride, silica gel plates) gave a new product (R _f = 0.73). The excess solvent was then removed, the dark red oil was flash chromatographed (silica gel, methylene chloride), the solvent removed in vacuo and the resulting light yellow oil purified by flash chromatography (silica gel, methylene chloride). 25 g (076 mol) of the title bromo ester are obtained in the form of a clear oil (75%) containing isopropanol solvate and containing less than 5% non-brominated ester.

1 H-NMR (CDCl 3):

8.16 (d, 2H), 7.38 (d, 2H), 5.05 (septet, 1H),

4.14 (dd, 1H), 2.88 (m, 4H), 2.39 (m, 4H), 1.29 (d, 6H);

@ 13 C-NMR (CDCl3):

168.7, 147.7, 129.3, 123.8, 69.9, 45.1, 35.6, 33.0,

21,5,21,2.

Example P)

a- [3- (4-Nitrophenyl) propyl] -1,4,7,10-tetraaza-cyclododecane-1-acetic acid methyl ester

Cyclenic free base (3.137 g, 18.2 mmol) is mixed with 30 ml of pentene-stabilized chloroform and 4.81 g (14.6 mmol) of d, l-2-bromo-4- (4-nitrophenyl) -butyric acid is added. isopropyl ester (prepared according to Example K) over 5 minutes under nitrogen with stirring. The resulting reaction mixture was then stirred at room temperature for 24 hours. TLC analysis (solvent system 2) gave the title monoalkylated product (R _f = 0.78, detection: ninhydrin, iodine, UV activity). The resulting yellow chloroform solution was purified by flash chromatography (silica gel column previously eluted with 5% methanol in chloroform) eluting with 300 mL of such solvent followed by solvent system 2, the fractions containing pure title product were combined and concentrated, yielding 4.85 g (5.46 mmol) of the title compound as the free base.

Yield: 79%.

1 H-NMR (CDCl 3):

8.15 (d, 2H), 7.40 (d, 2H), 5.07 (ρ, 1H), 3.35 (dd, 1H), 2.65-3.0 (m, 13H), 2 , 5-2.64 (m, 4H), 2.14 (m, 1H), 2.00 (m, 1H), 1.28 (dd, 6H);

@ 13 C-NMR (CDCl3):

171.6, 149.5, 146.5, 129.2, 123.6, 68.1, 62.7, 49.2,

47,5,45,9,45,7, 32,9,31,0,22,1,22,0;

IR (CDCl 3) cm -1:

3231 (N-H), 2978, 2933, 1721 (estercarbonyl), 1601, 1512, 1458, 1345, 1107;

FAB MS m / e 422 [M + H] +, 408, 392.

Preparation of the final product ligands

Example 1

α- (4-Nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid 1-monomethyl-4,7,10-triethyl ester was mixed with 4.6 g (33 g) of acetonitrile. 3 mmol) of freshly powdered anhydrous potassium carbonate and then dissolved in 700 mg (1.91 mmol) of α- (4-nitrophenyl) 1.4.7.10-tetraaza-cyclododecane-1-acetic acid-1-methyl ester [D] Prepared according to Example 1], and 1.022 g (6.12 mmol) of ethyl bromoacetate are added in one portion. After stirring for 4 hours at room temperature, the suspension was filtered in vacuo, the cake was washed twice with 15 mL of acetonitrile, and the filtrate was concentrated in vacuo at 38 ° C to give the title tetraester as a purple solid. The resulting tetraester is then purified by chromatography (silica gel, 5% C ₂ H ₅ OH / CHCl 3 followed by solvent system 3) to give 620 mg (0.988 mmol, 52%) of the desired product.

FABMS [M + H] + = 624.

1 H-NMR (CDCl 3):

8.24 (d), 7.45 (d), 4.94 (s), 4.35-4.10 (m), 3.79 (s), 3.75-1.84 (m), 1.34-1.22 (m);

C-NMR (CDCl 3):

174.3, 173.8, 173.6, 171.5, 147.6, 138.9, 131.5,

123.4, 64.8, 61.5, 61.4, 60.2, 55.4, 55.0, 53.1, 52.9,

52.7, 52.4, 51.9, 51.7,48,8,48,3,44,9,19,1,14,1.

Example 2

α- (4-Nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7.10-tetraacetic acid In a solution of 6N hydrochloric acid in 300 ml (0.478 mmol) of α- (4-nitrophenyl) -1 4,7,10-Tetraaza-cyclododecane-1,4,7.10-tetraacetic acid monomethyl triethyl ester (prepared as in Example 1) and the resulting mixture was refluxed overnight. At the end of the reflux, the solution was concentrated in vacuo at 70 ° C, the resulting solid was dissolved in water (3 mL), filtered and the filtrate was concentrated. This gave 253 mg (0.378 mmol, 79%) of crude product which was purified by preparative TLC chromatography (silica gel, developing 20% NH ₄ OAc / CH ₃ OH, w / v).

1 H-NMR (D ₂ O):

8.21 (d), 7.42 (d), 4.83 (s), 3.83-2.19 (m), 1.92 (s);

FAB MS = [M + H] ⁺ = 526.

13 C NMR (D ₂ O):

175.0, 173.6, 171.8, 167.0, 166.3, 146.9, 138.3,

130.1, 123.0, 62.2, 54.0, 53.0, 52.2, 50.4, 97.3, 46.8,

44.8, 42.8, 20.0.

Example 3

v- (4-Aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid; (BA-DOTA)

Method A)

The nitro group of tetraacetic acid (compound prepared in Example 2) is catalytically hydrogenated using an Adams catalyst (PtO ₂ ) and hydrogen

This gives the title product in essentially quantitative yield.

1 H-NMR (D ₂ O):

8.12 (d), 7.86 (d), 5.66 (s), 4.73-4.57 (m), 4.27-3.78 (m), 3.39-2.91 (m);

¹³ C-NRM (D ₂ O):

176.54, 176.52, 176.46, 141.1, 128.9, 120.9, 112.5, 65.0, 555.9, 55.7, 53.6, 49.7, 49, 5, 49.3, 45.7, 45.4,

44,9,44,6,41,4.

The product was also characterized by FAB analysis ([M + H] ⁺ = 496) and anion exchange HPLC (Q-Sepharose ™).

B) Method

Alternatively, the title compound is prepared by converting the monoester tetraamine compound of Example D into a monoacid tetraamine compound (6N hydrochloric acid, refluxing overnight) and then alkylating the product in aqueous medium using bromoacetic acid, followed by reducing the nitro group to an amine group (PtO ₂ catalyst) to yield a compound having the above physical constants.

Example 4

a- (4-Nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetic acid in 2.0 ml of 6N hydrochloric acid was dissolved in 2.0 g (5.48 mmol) of the monoester tetraamine compound [D ), the mixture was heated at 95 ° C for about 10 hours and then concentrated in vacuo at 75 ° C to give 2.53 g (5.11 mol, 96%) of the monoacet tetraamine tetrahydrochloride as a yellow solid. in the form of a solid.

The above monoacid tetraamine tetrahydrochloride (0.5 g, 1 mmol) was dissolved in 15 mL of water and adjusted to pH 9 with sodium hydroxide. A solution of bromoacetic acid was prepared separately (0.71 g, 5.13 mmol) and added to the above aqueous solution. The pH is again adjusted to 9 and maintained at this value by the addition of a small amount of 1N sodium hydroxide during the reaction. The reaction mixture was stirred and aliquots were removed at various times to detect the progress of the alkylation reaction by anion exchange HPLC. When the amount of dialkylated product (all three acetate groups present) has reached its maximum, the reaction product is lyophilized to give a dark solid. This crude mixture of alkylated products was then chromatographed on silica gel (solvent system 1) followed by preparative anion exchange chromatography (0-100% 1 mol NH ₄ OAc), then preparative TLC chromatography (development, solvent system 4), and finally silica gel chromatography (solvent system 4). ). This effective purification afforded 30 mg (0.06 mmol, 6%) of the title compound as a yellow solid.

FABMS: [M + H] + = 468.

• 1 H-NMR (D ₂ O):

8.12 (wide s), 7.35 (wide s), 4.76 (s), 3.57-3.44 (m), 2.97-1.83 (m), 1.80 (s) );

¹³ C NMR (D ₂ O):

181.75, 180.68, 179.00, 175.37, 147.70, 141.22, 133.08, 123.53, 68.44, 59.72, 56.00, 52.80, 49.96, 49.29, 47,86,45,36,44,26,42,78,42.25, 23.72.

Example 5

α- (4-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetic acid pentahydrochloride and α- (4-aminophenyl) -1,4,7,10 -tetraaza-cyclododecane-1,4,10-triacetic acid pentahydrochloride

In the same manner as in Example 4, a crude mixture of alkylated products was prepared and the resulting mixture was chromatographed on silica gel (20% w / v NH ₄ OAc / CH ₃ OH) and the appropriate fractions were combined and evaporated to dryness. The crude product, which contains a significant amount of NH ₄ OAc, was dissolved in 80 mL of NANOpure ™ water and lyophilized to give a light brown solid. This material was dissolved in 20 mL NANOpure ™ water, mixed with PtO ₂ catalyst and hydrogenated under hydrogen atmosphere until hydrogen uptake ceased. The catalyst was removed by filtration in vacuo, the filtrate was lyophilized, and the resulting yellow solid was dissolved in 3 mL of solvent system 4 and chromatographed on silica gel, eluting also with solvent system 4. The combined fractions were then evaporated in vacuo and the product was lyophilized to give 779.6 mg of a light yellow solid. The ¹³ C-NMR and proton NMR analysis carried out showed that the product obtained was a 50/50 mixture of the two possible geometric isomers. This isomeric mixture was mixed with excess hydrochloric acid and lyophilized to give 548.4 mg (61%) of the title compound, m.p.> 270 ° C. Anion exchange chromatography (HPLC, solvent system III) performed showed a major peak (> 94% purity), FAB MS: [M + H] + = 438.

Example 6

a- [2- (4-Nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid, 1,4,7,10-tetramethyl ester 1.43 g (3.63 moles) of α- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1-acetic acid 1-methyl ester [Example B] prepared] in acetonitrile, purged with argon gas (40 mL), anhydrous potassium carbonate (1.71 g, 12.34 mmol) was added with vigorous stirring, and methyl bromoacetate (1.78 g, 11.61 mmol) was added and the resulting mixture Stir at 25 ° C for 48 hours. TLC analysis gave a new product (R _f = 0.62, solvent system 2), to which 6 g of flash silica gel was added and the acetonitrile removed in a rotary evaporator. The residue was applied to a 1 x 16 inch silica gel column previously eluted with 5% methanol in chloroform. About 400 mL of the resulting solution was eluted several times to remove various non-polar impurities as well as unreacted alkylating agent. After applying the solvent system 2, the product-containing fractions (R _f = 0.62) were collected and concentrated to give 2.1 g (3.44 mmol, 95%) of the title compound as a yellow glass.

HU 219 485 Β ban. ¹ H NMR analysis showed the product to be a 2: 1 mixture of geometric isomers.

1 H-NMR (CDCl 3, 50 ° C):

8,137 (d), 8,128 (d), 7,398 (d), 7,385 (d), 3,82 (s), 3,76 (s), 3,75 (s), 3,74 (s), 3, 68 (s), 1.5-3.52 (m);

3 C NMR (CDCl 3, 50 ° C, 75 MHz):

175.8, 174.2, 174.1, 174.0, 149.0, 129.5, 129.3, 123.6, 60.1, 555.1, 52.8, 52.7, 52, 6, 52.4, 52.2, 52.1, 52.0, 51.2, 51.1, 51,0,49,1,48,8,47,5,45,2, 34,2, 32.6.

Example 7

а. - [2- (4-Amino-phenyl) -ethyl] -l, 4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1,4,7,1O tetramethyl

1.41 g (2.31 mol) of α- [2- (4-nitrophenyl) ethyl] 1.4.7.10-tetraaza-cyclododecane-1,4,7 (400 mg, 10% palladium on carbon) in 25 ml of methanol are dissolved in nitrogen. , 10 tetraecetsav1.4.7.10- tetramethyl ester (prepared as in Example 6), hydrogen gas was bubbled through the solution for in excess of 10 ⁵ Pa pressure for 3 hours. The TLC shows formation of a strongly ninhydrin positive material _(Rf = 0.62, solvent system 2). The methanol catalyst slurry was then filtered through Celite, and the filtrate was evaporated to give the title compound (1.21 g, 2.08 mmol, 91%) as a white glassy solid (2: 1 mixture of conformation isomers). The small amounts of conformation or geometric isomers were separated from the major isomers by flash chromatography (10% methanol in chloroform) to give the title compound as an off-white powder, m.p. 89-95 ° C.

1 H-NMR (CDCl 3, 50 ° C):

б, 94 (d), 6.89 (d), 6.69 (d), 6.66 (d), 3.91 (s), 3.80 (s), 3.78 (s), 3 , 74 (s), 3.73 (s), 3.72 (s), 3.71 (s), 1.5-3.5 (m), 3 C NMR (CDCl 3, 50 MHz):?

176.1, 174.0, 173.9, 172.2, 170.4, 169.6, 144.2,

144.1, 130.6, 130.5, 129.5, 129.1, 115.9, 115.8, 62.6,

58.7, 55.1, 54.3, 52.7, 52.5, 52.3, 52.2.52.1, 52.0, 51.9,

51.8, 51.7, 50.2, 50,0,47,3,44,7,32,8,31,8, 30,0,25,2;

FAB MS: m / e 602 [M + Na +], 618 [M + K + J +, [624 M + 2Na + -H +] ⁺ .

Example 8 α- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid; (PA-DOTA) Dissolve 1.5 g (2.59 mmol) of α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4 (1.59 mmol) in concentrated hydrochloric acid. 7,10-Tetraacetic acid-1,4,7,10-tetramethyl ester (crude mixture 2: 1 prepared in Example 7) and the reaction mixture was heated at reflux at about 105 ° C for 6 hours. TLC performed (solvent system

1) indicates the conversion of the ester to the title compound (R _f = 0.88) in the form of its hydrochloride salt (R _f = 0.43). Excess solvent was removed by rotary evaporation and the remaining white solid was dried. The title compound was obtained as the hydrochloride salt (1.6 g).

1 H-NMR (D ₂ O, pD = 1.0, 90 ° C):

7.48 (d), 7.42 (d), 28-4.3 (m), 2.23 (m), 2.03 (m);

3 C NMR (D ₂ O, pD = 1.0, 90 ° C, 75 MHz):

176.4, 174.9, 171.6, 144.7, 132.9, 132.8, 130.4,

125.7, 61.7, 56.8, 56.0, 53.7, 53.1, 51.6, 47.9, 34.3, 37.6.

FAB MS: m / e [MH] +, 546 [M + Na +] +, 568 [M + 2Na ⁺ -H ⁺ ] ⁺ .

Flash chromatography and solvent system 1 removal of all traces of the ester can be accomplished by adding 1.1 g of the title product as the hydrochloride salt to the flash chromatography column. Fractions containing the title compound were collected as a mixed ammonium potassium salt (580 mg).

HPLC analysis showed material purity greater than 98% at 230 and 254 nm.

FAB MS: m / e 562 (M + K) ⁺ , 584 [M + K ⁺ + Na ⁺ -H +], 600 [M + 2K + -H + J +].

Example 9

Isolation of .- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acid, mixed potassium and ammonium salts

2.05 g of a mixture of tetra and trisacids (both a

Prepared according to the procedure of Example 8), dissolved in a minimum amount of solvent system 1, and then applied to a flash column of silica gel (38x7 cm) which had been previously eluted with the solvent. Fractions containing the title compound (R _f = 0.63, solvent system 1) were collected and contained 200 mg of the title compound mixture. According to * H- and ^{8 * * * * 13} C-NMR studies, the trisacid is a symmetric isomer (1,4,10-trisacid).

1 H-NMR (D ₂ O, pD = 0.5 with DCl, T = 90 ° C):

7.52 (d), 7.46 (d), 3.60 (m), 3.54 (m), 3.19 (m);

³ C NMR (D ₂ O, pD = 0.5, T = 90 ° C):

176.1, 170.2, 140.0, 132.7, 131.1, 126.1, 57.6, 57.2,

56,1,54,9, 53,2,51,5,51,2,31,2.

FAB MS: m / e 466 [M + H +] + 488 [M + Na ⁺ ] +, 504 [M + K + J +, 526 [M + K + Na +, - H ⁺ ] +, 542 [M + + 2K]. -H)] +.

Example 10

a.- (2-Methoxy-5-nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid tetramethyl ester 565 μΐ (5.97 mmol) methyl- bromoacetate was stirred

580 mg (1.47 mmol) of methyl α- (2-methoxy-5-nitrophenyl) 1,4,7,10-tetraaza-cyclododecane-1-acetic acid (prepared according to Example N) and 1.15. g (8.32 mmol) of powdered potassium carbonate in 20 mL of acetonitrile. The resulting mixture was stirred at room temperature for 2 hours and then filtered, the filtrate was concentrated in vacuo and the oily residue was chromatographed (silica gel, 8% methanol in methylene chloride). The title compound was obtained as a yellow solid (469 mg, 52%), TLC, R _f = 0.4 (silica gel, 10% CH ₃ OH in CHCl ₃ ).

3C-NMR (CDCl3):

HU 219 485 Β

173.2, 172.6, 161.2, 139.1, 125.1, 124.6, 120.5,

110.7, 57.0, 55.6, 53.5, 52.6, 51.3, 50.8, 46.8, 46.0, 44.0.

Example 11

a- (2-Methoxy-5-nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid Example 10 was dissolved in concentrated hydrochloric acid (5 mL, JT). Baker ULTREX) and the resulting solution was refluxed under nitrogen for 5 hours. The solution was then evaporated to dryness in vacuo and the resulting solid was chromatographed (silica gel, solvent system 6) to give 209 mg of the title compound as an off-white solid. The material was converted to the tetrahydrochloride salt.

³ C-NMR (D ₂ O-DC 1, 80 ° C):

171.0, 166.9, 161.2, 139.0, 125.5, 119.3, 110.7,

56.8, 54.5, 52.7, 51,0,49,2,49,0,46,3,42,9.

Example 12 ct .- (2-Methoxy-5-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid, tetraammonium salt 157 mg of the compound obtained in Example 11 are dissolved. ml of water was added 20 mg platinum oxide and the mixture hydrogenated at room temperature for 1 hour at 10 ⁵ Pa pressure. The mixture was then filtered and the filtrate was chromatographed (silica gel, solvent system) to give 141 mg of the title compound as an off-white solid.

• ³ C-NMR (D ₂ O-DC 1):

175,5,172,6,172,3,159,9,128,8,127,5,124,6,123,8,

115.5, 61.2, 58.1, 57.3, 55.7, 53.4,51,6,49,6,47.4.

Example 14 1- (2-Methoxy-5-nitrobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid, 4,7,10-trimethyl ester in 20 ml of acetonitrile mixed with 1 g of 1- (2-methoxy-5-nitrobenzyl) -1,4,7,10-tetraaza-cyclododecane (prepared according to Example E) and then 1.6 g (11 mmol) of methyl bromo are added with stirring. acetate in one installment. After 1 hour, 0.3 g of potassium carbonate was continuously stirred at room temperature over 12 hours, the mixture was filtered, the filtrate was concentrated in vacuo and the residue was subjected to column chromatography (silica gel, CH ₃ CN / CH ₃ OH = 9: 1, v / v). v / v) to give the title triester as a white solid in 68% yield.

1 H-NMR (CDCl 3):

8.19 (d), 8.14 (s), 7.18 (d), 2.58-3.99 (m);

³ C-NMR (CDCl ₃ ):

173.53, 172.83, 164.59, 142.28, 128.51, 127.69, 126.49, 112.30, 57.25, 56.90, 55.03, 54.21, 53, 06, 52.09, 52.0, 51.54, 50.68, 50.30, 49.85, 49.63, 49.31, 49.00,48,68,48,37,48.05, 47,70,47,39.

Example 75

- (2-Methoxy-5-nitrobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid dissolved in 6 M hydrochloric acid (0.25 g, 0.45 mmol) and stirred at 100 ° C for 24 hours. The reaction mixture was then cooled to room temperature, water (5 mL) was added and the resulting aqueous solution was freeze-dried. The beige solid thus obtained was subjected to column chromatography (silica gel, solvent system 5) to give the desired trisacid in 60% yield as an off-white solid, m.p. 228-230 ° C (dec.).

• 1 H-NMR (D ₂ O):

8.18 (d), 8.09 (s), 7.12 (d), 3.87 (s), 2.10-3.60 (m);

³ C NMR (D ₂ O):

180.45, 179.80, 163.66, 140.22, 128.60, 126.24, 122.66, 111.50, 59.91, 59.06, 56.44, 52.75, 50, 92,48,84.

Example 16

- (2-Methoxy-5-aminobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid in ml of an aqueous solution of 50 mg of PtO ₂ and 50 mg of the preceding, 15. The resulting solution was filtered, freeze-dried to obtain the desired aniline derivative as a brownish solid (95%), m.p.> 240. ° C (dec.).

• 1 H-NMR (D ₂ O):

7.54 (s), 7.21 (d), 7.09 (d), 7.05 (s), 6.88 (s), 3.84 (s), 3.78 (s), 2.85-3.56 (m);

³ C-NMR (D ₂ O):

180.59, 179.77, 153.38, 124.16, 124.03, 122.82, 120.18, 113.68, 112.43, 59.06, 57.02, 55.96, 53, 67 ,. 50.52, 50.08, 49.70, 49.04, 48.32.

Example 17 1- (2-Hydroxy-5-nitrobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid

1- (2-Hydroxy-5-nitrobenzyl-1,4,7,10-tetraaza-cyclododecane (prepared in Example F)) (200 mg, 0.62 mmol) was dissolved in water (1 mL), and 309 mg (2.2 mmol) was added. Bromoacetic acid and 3.5 mL (about 1 mol) of sodium hydroxide with stirring at room temperature. The reaction was monitored by LC chromatography (anion exchange, Q-Sepharose ™), while maintaining the pH at about 8 by adding sodium hydroxide as needed. After 12 hours, the resulting solution was freeze-dried and the solid was subjected to column chromatography (silica gel, solvent system 5). The major yellow fraction was concentrated, dissolved in water and freeze dried to give the desired product as a light yellow powder (150 mg, 49%), mp 230-235 ° C (dec.).

³ C-NMR (D ₂ O):

166.41, 165.60, 160.00, 119.91, 116.02, 113.85, 111.71, 1044.59, 45.20.20.44, 50.43, 96, 36.43.

Example 18 1- (2-Hydroxy-5-aminobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid dissolved in 200 mg (0.4 mmol) of water in ml Purify with argon

After addition of 30 mg of PtO ₂ , the mixture is hydrogenated on a Parr apparatus. After the yellow had completely disappeared, the solution was flushed with argon, filtered, and the aqueous solution was freeze-dried to yield 146 mg (78%) of the title compound as a brownish solid.

Example 19 1a - [2- (4-Nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1- (R, S-acetic acid) -4,7,10-tris [ (R) -v.-acetic acid methyl] -l-isopropyl-4,7,1O trimethyl ester; (PN-DOTMA trimethyl isopropyl ester)

100 g (2.37 mmol) of α- [3- (4-nitrophenyl) propyl] 1,4,7,10-tetraaza-cyclododecane-1-acetic acid-1-methyl ester (prepared according to Example P) dissolved in 35 ml of anhydrous acetonitrile, 1.15 g (8.32 mmol) of anhydrous potassium carbonate are added under vigorous stirring under a nitrogen atmosphere, followed by addition of the optically active α-benzenesulfonate derivative of lactic acid methyl ester (1.79 g, 7.39). mmol, S: R = 98.2) and the resulting mixture was stirred at room temperature for 60 minutes. TLC analysis shows the formation of a new product (R _f = 0.71, solvent system 2 for the title tetraester and R _f = 0.89 for the triester). The resulting solution was mixed with the suspended carbonate salt in 40 ml of chloroform and the precipitated inorganic salt was filtered off. The solvent was removed from the filtrate and the resulting crude orange oil was chromatographed twice on a 1 x 16 inch flash silica gel column using 2 solvent systems. In this way, the title compound is obtained as a non-resolved mixture of the tetraester diastereomers (1.00 g, 1.47 mmol, 62%), which does not contain a lower alkylated product.

This material contains about 0.5 equivalents of unreacted benzene sulfonate, according to Ή NMR analysis. The NMR spectrum of the material at 60 ° C was not consistent with chloroform.

1 H-NMR (CDCl 3, 60 ° C):

7.9-8.1 (m, 2H), 7.2-7.4 (m, 2H), 5.06 (m, 1H), 3.4-4.0 (m, 9H), 1, 7-3.4 (m, 20H), 1.0-1.7 (m, 15H);

IR (CDCl 3) cm -1:

2980, 2840, 1725, 1710 (ester carbonyl), 1590, 1510, 1440, 1340;

FAB MS m / e 702 (M + Na +) ⁺ , 687.

Example 20 1a- [2- (4-Aminophenyl) ethyl] -1,4,7,10- [tetraaza-cyclododecane-1- (R, S-acetic acid) -4,7,10-tris] [(R) -S-acetic acid methyl] -l-isopropyl-4,7,10-trimethyl ester;

(PA-DOTMA Trimethyl Isopropyl Ester)

950 mg (1.4 mmol, uncorrected) of the compound prepared in Example 19 which is unreacted α- [3- (4-nitrophenyl) propyl] -1,4,7,10-tetraaza-cyclododecane-1-one. acetic acid 1-methyl ester, dissolved in 10 ml of 30% aqueous methanol containing 1 g of 10% palladium on carbon under a nitrogen atmosphere, followed by excess hydrogen gas for 7 hours. TLC was then performed which showed the formation of a highly positive material for ninhydrin (R _f = 0.62, solvent system 2, R _f = 0.71 for starting material). The methanolic catalyst slurry was then separated by filtration through Celite and the solvent was evaporated to yield 800 mg of crude title compound as a clear oil containing benzenesulfonic acid (R _f = 0.09, solvent system 2), the sulfonate ester hidrogenolíziséből inherently. The crude oil is then chromatographed on a 1 x 8 inch flash silica gel column eluting the pale yellow bands. The title product was eluted with solvent system 2 (480 mg, 52%) and a mixture of non-resolved diastereomers and geometric isomers. Three different b-quartets were observed in the 1 H-NMR spectrum in the aromatic region:

1 H-NMR (CDCl 3, 30 ° C):

6.63-8.0 (m (quartets, 4H), 5.07 (m, 1H, isopropylmethine H), 3.53-3.9 (m, 13H 33 singlet: 3.85, 3, 73, 3.69 and 3.56 3.46 (m, 1H), 3.25 (broad t, 3H), 2-3.1 (m, 14H), 1.0-2.0 (m, 15H);

¹³ C-NMR (CDCl 3, 30 ° C):

177.3, 177, 176.4, 176.2, 175.9, 173.4, 172.9,

170.6, 145.3, 144.9, 130.8, 129.7, 129.3, 129.1, 128.8,

128.4, 127.5, 126.3, 115.2, 115.1, 114.9, 69.0, 68.4,

67,3,61,6,57,8,57,4,56,7, 56,5, 56,4,52,9, 52,7, 52,1,

50.8, 50.7, 50.6, 47.3, 47.1 47.0, 46.9,46.5, 46.3, 46.1,

44.8, 44.5,44.3, 34.1, 32.9, 32.5, 32.2, 31.8, 24.8, 22.2,

22.1, 22.21,8,21,6,15,8,14,9, 7,7, 7,5, 7,4, 7,1;

IR (CDCl 3) cm -1:

2,960,2840,1720,1510,1450,1375;

FAB MS: m / e 672 [M + Na ⁺ ] +, 686.

Example 21 1a - [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1- (R, S-acetic acid) -4,7,10-tris- [(R) -a.-methyl acetic acid];

(PA-DOTMA)

The diastereomeric mixture of Example 20 (400 mg, 0.59 mmol) was dissolved in 50 mL of 6N hydrochloric acid and heated at reflux for 30 hours. TLC analysis (solvent system 1) showed the conversion of the ester (R _f = 0.98, solvent system 1) to a new product with two new spots (R _f = 0.6, higher diastereomer, R _f = 0.54, low diastereomer, solvent system 1, both spots UV, ninhydrin and iodine positive). Excess solvent was removed in a rotary evaporator and dried to give the crude diastereomeric mixture of the title compounds (403 mg) as the hydrochloride salt. Preparative separation of the two diastereomers was accomplished by adding 290 mg (0.51 mmol) of the crude salt to a 1 x 13 cm silica gel flash column and eluting with 8 solvent systems. The higher R _r diastereomer (119 mg, 0.21 mmol) is swallowed in a purer form than the lower R _r diastereomer (90 mg, 0.16 mmol), since the higher R _r diastereomer is first eluted from the column.

Ή-NMR for diastereomers with higher R _r values:

1 H-NMR (D ₂ O, 90 ° C, pp = 7.5):

7.12 (d 2H), 6.80 (d, 2H), 3.66 (m, 1H), 3.57 (broad t, 3H), 2 to 3.4.4 (m, 19H), 1.89 (m, 1H), 1.37 (m, 1H),

1.15 (d, 3H), 1.10 (d, 3H), 1.06 (d, 3H);

HU 219,485 Β • 3 C NMR (D ₂ O, 90 ° C, pp = 7.5):

184.4, 184.2, 184.0, 183.3, 135.9, 132.6, 131.7,

119.1, 78.2, 69.0, 61.7, 61.5, 61.1, 57.4, 54.1, 49.7, 49,6,48,2,47,7,47, 2, 34.8, 33.8, 9.6, 9.1, 9.0;

FAB MS: m / e 566 [M + H + J +, 588 (M + Na ⁺ ] +, 604 (M + K +] ⁺ , 626 [M + K + + Na + -H +] +, 642 (M + 2K + -). H + J +.

Finished products - preparation of complexes

The metal-ligand complexes were prepared by various methods. In these methods, the metal and ligand were mixed in an aqueous solution while adjusting the pH to the desired value. The complexation was carried out in a solution containing salts and / or buffers and water. Occasionally, the solutions were heated to obtain a higher complex yield than when the complexation was carried out at ambient temperature. It was also found that the processing step in the synthesis of BFC compounds also influences complexation.

Example 22

a - (4-Aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid ammonium salt samarium (III);

Sm (BA-DOTA) mg (0.094 mmol) of α- (4-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid (prepared as in Example 3) is dissolved. 10 L of water and mixed with 3 mL of 0.043 M Sm (OAc) ₃ (48.8 mg, 0.128 mmol) at pH 6-7. The resulting solution was then heated to 100 ° C and the degree of complexation was determined by anion exchange HPLC. At this point, the complexation followed by anion exchange chromatography (HPLC System III) was completed, the solution of the complex cooled to room temperature and freeze-dried. The samarium complex was purified by silica gel chromatography (solvent system 1), whereby the title compound was swallowed with 82% yield. The resulting complex was identified by TLC, FAB MS, and anion exchange HPLC.

• H (D ₂ O):

8.94, 7.81, 7.21, 6,97,6,80, 5.63, 5,01,4,58,4,01, 3,56, 1,78,1,53,1, 24.1,10,0.77, -2.80, -2.95, -3.21, -3.91;

¹³ C NMR (D ₂ O):

190.3, 184.9, 181.4, 146.8, 143.3, 122.7, 116.4,

80.8, 72.6, 64.2, 57.2, 55.8, 54.7, 53.4, 51.5,49,7,45,5,

43,5,23,6.

FABMS: [M + H ⁺ ] ⁺ = 645 (Sm isotope).

Example 23

α- (4-Aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid samarium (III) complex

150 µl of Sm-153 solution (3 x 10 ~ ⁴ moles in 0.1 N hydrochloric acid) and 600 µΐ SmCl ₃ ( ₃ x 10 ~ ⁴ moles) were mixed with 0.1 N hydrochloric acid and 5 µΐ 2 M NaOAc was added followed by 45 µΐ 50 mM ligand (prepared as in Example 3) in HEPES buffer. The pH of the solution was adjusted to 7 using 275 μΐ 0.5 M HEPES buffer. This solution was separated into two fractions of 500-500 μΐ and one fraction was heated at 100 ° C for 1 hour.

The solutions are then passed through a SP-Sephadex ™ cation exchange resin, and the percentage of metal in complex form is determined as described above. According to the result, 99.6% of the metal is complexed in the heated sample and 96.6% in the unheated sample.

Stability of chelates as a function of pH

The stability of the metal-ligand complexes at different pH values was determined by determining the amount of complexes in solution. At low pH, metal release can occur due to protonation of the ligand. At high pH, metal hydroxides are also involved in chelation. Therefore, when researching inert chelates, it is necessary that the inert chelate remain stable at both high and low pH.

The stability of the chelates of the invention as a function of pH was tested according to the methods described in the following examples. In some cases, the uncomplexed metal was removed by passing the solution through a cation exchange resin. Dilute sodium hydroxide and hydrochloric acid were used to adjust the pH of the complex solutions to between 1 and 14. The metal content of the complex was determined according to the methods described above.

Similarly, bifunctional chelate conjugates were prepared and tested at pH 2.8.4 and 6.0. The amount of conjugate remaining in the solution was determined by HPLC using a radiometric detector. The results obtained are summarized in Example XX of the following biological section.

Example 24

stability of the complex of .- (4-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium (III) as a function of pH

The heated and non-heated samples of Example 22 were aliquoted into 2 x 250 μΐ aliquots. The pH of one of the 250 μΐ samples was adjusted to the values summarized in the following table using hydrochloric acid. The second 250 μΐ sample was adjusted to the desired pH with sodium hydroxide. When the sample reached the desired pH, 25 μΐ aliquots were removed and the metal content of the complex determined as described above. The results are summarized in the following table:

PH Complex% heat treated Not snow treated 1 98.9 92.7 3 99.6 93.3 5 99.6 92.9 7 99.6 95.7 9 99.9 95.8 11 99.6 95.7 13 99.6 94.5

HU 219 485 Β

The data in the table show that the complexes of the present invention are stable, regardless of heating or pH.

Example 25

u. - (4-Aminophenyl) -1,4,7,10-tetraaza-cyclododecane1.4.7.10-tetraacetic acid-yttrium (III) complex μΐ 1.4 mg / 100 μΐ a- (4-aminophenyl) -1 A solution of 4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid (prepared as in Example 3) was added to 100 μΐ of a 0.03 M solution of Y (OAc) ₃ containing Υ-90 contain. To the resulting solution was then added 500 μΐ of water followed by 125 μΐ of 0.5 M NaOAc followed by 265 μΐ of water to make up the total volume to 1 ml. The resulting solution was then divided into two portions. One portion was heated at 100 ° C for 1 hour, and then the metal content of the complex was determined in both heated and unheated samples by the cation exchange method described above. According to the results, 84% of the metal contained the heated and unheated samples.

Example 26 α- (4-Aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7.10-tetraacetic acid-lutetium (III) complex

A solution of α- (4-aminophenyl) 1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid prepared in Example 3 was prepared in distilled water at a concentration of 1.63 mg / ml.

A solution of lutetium chloride in 0.1 N hydrochloric acid was prepared at a concentration of 3.9 mg / ml (0.01 mol). Lutetium 177 (0.3 mmol) was used as a label.

μΐ 0.01 M lutetium solution was added to 2 μΐ Lu-177 solution. The ligand prepared above was then added and the volume was adjusted to 1 mL with HEPES buffer (0.1 mol, pH 7.6). The resulting solution was 0.03 mM for ligand and lutetium. The resulting solution was then heated at 100 ° C for 1 hour and the Lu content was determined using the cation exchange method described above at 86%.

Example 27

v. - (4-Isothiocyanato-phenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid samarium (III) Compound mg (10.8 pmol) (4-Aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium (III) is dissolved in 400 µl of water, 50 µl of thiophosgene is added, followed by 400 µl of CHCl ₃ , and the resulting biphasic mixture is stirred vigorously for 30 minutes. The aqueous phase is then extracted four times with 500 µl of CHCl ₃ , and the aqueous phase is lyophilized to give the title compound in quantitative yield.

The compound prepared as above has bands at 272 nm and 282 nm. Based on TLC analysis (silica gel, 75: 25-CH ₃ CN: H ₂ O, v / v) _Rf value of 0.38, 0.19 Rf in the starting material.

IR: (-SCN) = - 2100 cm -1;

FABMS: [M + H +] + = 687.

Example 28

а. - (4-Aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-ammonium salt-yttrium (III) complex mg (160 mmol) of the α- (4-amino) prepared according to Example 3. Phenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid is dissolved in 1 ml of water and 3 ml of an aqueous solution of Y (OAc) ₃ containing 51 mg (192 mmol) are added. The resulting mixture was then adjusted to pH 6-7 and heated at 100 ° C for 7 hours. The resulting solution was then filtered through glass wool and lyophilized to give 126 mg of a yellow solid. This material was chromatographed on silica gel (solvent system 1) to give 71 mg (76%) of the desired complex product. The anion exchange analysis carried out showed that the retention time of the product obtained was the same as that of the Sm complex.

13 C NMR (D ₂ O):

180.8, 180.5, 179.9, 146.1, 133.4, 122.0, 116.1,

74.9, 66.3, 56.3, 55.8, 55.4, 55.1, 54.8, 52.2, 46.0, 44.0, 23.6;

• 1 H-NMR (D ₂ O):

б, 90 (d), 6.70 (d), 4.40 (s), 3.45-3.06 (m), 2.71-2.10 (m), 1.75 (s).

FABMS: [M + H +] + = 582.

Example 29 Sodium Salt Yttrium (III) Compound α- (4-Isothiocyanato-phenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid Compound 28 (17 pmol) Example 28 The resulting compound was dissolved in 400 µl of water, then 64 µl of thiophosgene (excess) and 400 µl of CHCl ₃ were added and the resulting mixture was stirred vigorously for 40 minutes. A small amount of solid sodium bicarbonate was added several times during this time to maintain the pH at about 8. After completion of the reaction, the aqueous layer was separated, extracted four times with 1 mL of CHCl ₃ and lyophilized. The title compound was identified by TLC and UV spectroscopy.

Example 30

a- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid yttrium (IIl) complex ammonium salt;

NH ₄ [Y (PA-DOTA) J

The α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid ligand prepared according to Example 8 is converted into a mixed protonated ammonium salt using a highly cationic ion exchange resin (SP-Sephadex ™ C-25, Pharmacia). The column is then washed with distilled water containing the protonated form material, then the concentrated aqueous column of ligand is discarded and then washed with distilled water. The ligand was eluted from the column with 0.5 M ammonium hydroxide. The eluent is used in anhydrous form.

HU 219 485 Β

Y (OAc) ₃ -4H ₂ O (0.0728 g, 0.215 mmol) was dissolved in distilled water (5 mL) and added to the stirred protonated ammonium salt (0.120 g, 0.215 mmol), which was dissolved in water (5 mL). The resulting solution was then heated to reflux and adjusted to pH 7 with 0.1 M ammonium hydroxide. After refluxing for 1 hour, the solution was cooled and evaporated to dryness. Excess NH ₄ OAc was removed by heating and the remaining white solid was heated in an oil bath at 105 ° C under vacuum. This gives 0.142 g (94%) of the title compound containing 1 equivalent of ammonium acetate.

¹³ C-NMR (D ₂ O, 88 ° C, 75 MHz):

23.8, 27.6, 35.4, 49.6, 55.0, 56.2, 56.9, 57.1, 65.7, 68,0,121,7,132,6,138,8,180,7,181,4,182, 0,183,1;

FAB MS: m / e 610 (positive ion, [M - + 2H ⁺ ] ⁺ ), 608 (negative ion, M -).

Example 31

v. Ammonium Compound of [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid, samarium (III);

NH ₄ [Sm (PA-DOTA) J

Example 30 was repeated except that Sm (OAc) ₃ -3H ₂ O (0.082, 0.215 mmol) was used in place of Y (OAc) ₃ -4H ₂ O. In this way, the title compound (0.155 g, 94%) containing 1 equivalent of ammonium acetate was obtained.

¹³ C-NMR (D ₂ O, 88 ° C, 75 MHz):

23.5, 27.2, 35.6, 50.5, 54.5, 56.0, 57.2, 57.9, 69.5, 71.5, 122.3, 133.0, 139, 7, 181.2, 188.4, 189.3, 190.9.

FAB MS: m / e 673 (positive ion, [M - + 2H ⁺ ] ⁺ , Smisotope), m / e 671 (negative ion, M -, Sm - isotope).

Example 32

a. ammonium salt of .- [2- (4-Isothiocyanotophenyl) ethyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium (III);

NH ₄ [Sm (SCN-PA-DOTA)]

27.9 mg (381.56 g / mol, 73.1 pmol) of Sm (OAc) ₃ -3H ₂ O are dissolved in 10 ml of distilled water, mixed with 42 mg of PA-DOTA dissolved in 10 ml of distilled water, mixed with ammonium potassium salt. (632.97 g / mol, 66.4 pmol) and the resulting mixture was heated on a steam bath. After 30 minutes, the reaction was complete (Sm (PA-DOTA) formed by HPLC analysis). The solution was dissolved in 10 ml of chloroform elkeveqük CsCl 45.4 mg (114.98 g / mol, 395 pmol) and extracted with _two funnel. The reaction is complete in about 1 minute (HPLC analysis), no positive ninhydrin reaction (0.2t in ethanol) on silica gel TLC plate, showing initial [SM (PA-DOTA)] chelate positive ninhydrin reaction. The chloroform layer was removed, the aqueous layer was washed with chloroform (2 x 20 mL), and the aqueous layer was evaporated to dryness by addition of acetonitrile (100 mL) and evaporation at ambient temperature under a stream of nitrogen to give 56 mg of the title compound.

FAB MS: m / e 715 (positive ion, [M + 2H ⁺ ] ⁺ , Sm isotope), m / e 713 (negative ion, M, Sm isotope).

Example 33 The rate of chelation of y ^{+ J with} pA-DOTA

As determined by HPLC analysis

The rate of chelation of PA-DOTA with the Y ³ + ion was investigated as a function of the processing used to produce the PA-DOTA product. The chelation outcome was monitored by HPLC using an Alltech Econosphere ™ Cl8 100 mm column. The following gradients were used: A) 95: 5 pH 6.0, 0.05m NaOAc, buffer: CH ₃ CN, B) 30:70, pH 6.0, 0.05m NaOAc, buffer: CH ₃ CN, AB, 15 minutes. The PA-DOTA hydrochloride salt (retention time 1.31 min) and the PA-DOTA mixed ammonium potassium salt (retention time = 4.55 min), prepared according to Example 8, were used as a 1.6 M solution, pH = 6, 0.5 mol NaOAc buffer. 1 ml of each solution was reacted with 0.2 ml (8 pmol) of A (OAc) ₃ -4H ₂ O solution (40 mmol, pH 6.0, 0.5 mol NaOAc buffer). . Chelation was determined by the peak at 3.96 minutes _Rf value (compared to the sample of Example 30). The results of chelation are summarized in the following table.

PA-DOTA salt Complex% Time (minutes / temperature) PA-DOTA, > 90 <1 / room temperature HCl salt PA-DOTA blended <10 15 / RT ammonium potassium > 85 10/90 ° C

The table shows that the BFC purification method has an effect on the degree of chelation.

Example 34 1a- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecone-1- (R, S) -acetic acid 4,7,10-tris (methyl) -acetic acid) -ammonium complex (III), ammonium salt; NH ₄ [Sm (PA-DOTMA)] mg Sm (OAc) ₃ -3H ₂ O dissolved in 2 ml distilled water and 2 ml distilled water 30 mg a- [2- (4-aminophenyl) ethyl] -1,4 7,10-Tetraaza-cyclododecane-1 (R, S) -4,7,10-tris (methyl acetate) acetic acid (higher Rf diastereomer prepared in Example 21). The pH 6.5 solution was then heated on a steam bath and the reaction was monitored by HPLC. After heating for 30 minutes, the solution is concentrated to dryness under vacuum on a rotary evaporator.

FAB MS: m / e 715 (positive ion, [M + 2H ⁺ ] ⁺ , Smisotope), 737 (positive ion, [M + H ⁺ + Na ⁺ ] ⁺ , Sm-isotope), 713 (negative ion, [M ~], Sm isotope).

In the following examples, complex solutions were passed through an ion exchange column to remove excess free metal. The unstable systems would be re-equilibrated and a similar amount of free metal would be present in the solution. Inert systems are unbalanced and the amount of non-chelated metal present must decrease. In this way, even if the complex was formed with low yield,

21 purification, i.e. passage through the ion exchange column, results in a sufficiently stable complex without the presence of uncomplexed metal.

Example 35 α- (2-Methoxy-5-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid samarium (HI) complex

5.8 mg of α- (2-methoxy-5-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid prepared in Example 12 are dissolved in 325 μΐ NANOpure ™ water (3 x 10l ^2). M solution) and the resulting solution, 10 μΐ was taken and mixed with 990 μΐ 3χ10 ~ ⁴ M SmCl ₃ -oldattal comprising at SM-153 (0.1 n HCl). The pH was then adjusted to 7 with sodium hydroxide, the solution was divided into 500 μ 500 fractions twice, and one fraction was heated at 100 ° C for 1 h. The percentage of metal in both the heat-treated and non-heat-treated samples is then determined as a complex by the cation exchange method described above. The solution is then passed through a SP-Sephadex ™ resin and complexing is repeatedly determined on the purified samples. The results are summarized in the following table.

Example 37 α- (2-Methoxy-5-nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium (III) complex 7.9 mg 11. a- (2-methoxy-5-nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid prepared according to example 1d is dissolved in 464 μΐ NANOpure ™ water (3 x 10 ~ ² molar solution), 10 μΐ of the resulting solution was mixed with 950 μΐ 3x10 ⁴ M SmCl ₃ (containing Sm-153) in 0.1 N hydrochloric acid and adjusted to pH 7 with sodium hydroxide. The resulting solution was split into 2 χ 500 μΐ fractions, one of which was heated at 100 ° C for 1 h. The complexed metal is then determined in both heat-treated and non-heat-treated samples by the cation exchange method described above. The solutions were then passed through SpSephadex ™ resin and the amount of complexes in the purified samples was determined as above. The results are summarized in the following table.

Heat treated Not heat treated

Purified No Purified Not Purified Purified

Heat treated Not heat treated

Complex ⁰ / »100 72 100 46

Purified No Purified Not Purified Purified

Complex ⁰ / »96 95 97 89

Example 36

stability of the complex of .- (2-Methoxy-5-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium (III) as a function of pH Divide each of the heat-treated and non-heat-treated samples according to Figures 2 to 2 χ 250 μΐ. The pH of one aliquot is adjusted with hydrochloric acid and the other with sodium hydroxide according to the following table. After the samples have reached the desired pH, they are allowed to stand for 10 minutes and the amount of metal present in the complex is determined by the cation exchange method described above. The results are summarized in the following table.

Complex ⁰ / »

pH heat treated No heat 2 95 94 3 97 95 5 98 97 7 96 97 9 98 96 11 97 98 13 100 99

Example 38

stability of the .- (2-Methoxy-5-nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-Sm (III) complex as a function of pH

Each of the heat treated and untreated purified samples of Example 37 was divided into 2 aliquots. Adjust the pH of one aliquot with dilute hydrochloric acid and the other with sodium hydroxide as shown in the following table. After the samples have reached the desired pH, they are allowed to stand for 10 minutes and the metal content of the complexes is determined by the cation exchange method described above. The results are summarized in the following table.

Cleaned

pH heat treated Not heat-treated 2 99 99 4 100 100 5 100 100 7 100 100 9 100 100 11 100 100 13 100 100

Example 39

α- (4-Nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetic acid samarium (III) Compound 0.0219 mol (100 μΐ, 2.195 mmol) prepared according to Example 4. a- (4-nitrophenyl) -1,4,7,10-tetraazacyclododecane ciklodo21

The solution obtained from decane-1,4,7-triacetic acid was mixed with 0.043 M (25 μΐ, 1.05 mmol) of Sm (OAc) ₃ . The resulting mixture was subjected to anion exchange HPLC (Q-Sepharose ™ column, 1 x 25 cm, flow rate 2 ml / min, elution with 0-1 M NH ₄ OAc gradient over 30 min). After 1 hour, the complex is formed _(Rf = ll, l min), 77%, and the complex is separated from the fully ligandumcsúcstól (retention time = 15.5 min). Further evidence is that the title complex is formed by silica gel plate TLC analysis (solvent system 4) with ligand R _f = 0.59 and complex complex R _f = 0.00.

Example 40 α- (4-Aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetic acid-samarium (IIl) complex and α- (4-aminophenyl) -1,4 7,10-tetraaza-cyclododecane-1,4,10-triacetic acid-samarium (III) complex

A 0.022 molar solution of the isomers prepared in Example 5 (100 μΐ, 2.2 pmol) was contacted with a 0.043 mol (6.4 μΐ, 0.275 pmol) solution of Sm (OAc) ₃ . The resulting reaction mixture was then subjected to HPLC anion exchange assay (Q-Sepharose ™ column, 1 x 25 cm, flow rate 2 mL / min, elution with 0-0.25 M NH ₄ OAC, 300 min). After 40 minutes, the complex is formed (retention time = 9 minutes) in 66% yield and completely detached from the ligand peak (retention time 18.5 minutes).

Example 41 (t- (4-Aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetic acid-samarium-153 complex and α- (4-aminophenyl) -1,4,7; 10-Tetraaza-cyclododecem-1,4,10-triacetic acid-samarium-153 complex

Prepared according to Example 5 from α- (4-aminophenyl) 1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetic acid pentahydrochloride and α- (4-aminophenyl) -1,4 From a solution of 7,10-tetraazacyclododecane-1,4,10-triacetic acid pentachloride, a solution of ³ x 10 ~ ² mol was prepared by dissolving 4.2 mg of the substance in 300 μΐ NANOpure ™ water. A 10 μΐ aliquot of the solution was added to 15 µl of a 2 x 10 ^{2 2} M solution of SmCl ₃ (containing Sm-153) in 0.1 N hydrochloric acid, and the volume was made up to 1 mL with water. The pH was then adjusted to 7 with sodium hydroxide, the solution was divided into 500-500 μΐ fractions, and one fraction was heated at 100 ° C for 1 hour.

The metal content of the complexes is determined for both the cured and non-cured samples by the cation exchange methods described above. According to the results, 89% and 96%, respectively, of the metal contents were present in the heat-treated and non-heat-treated samples. ⁴²

Example 42 α- (4-Aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetic acid-samarium (III) complex and α- (4-aminophenyl) -1,4 Stability of 7,10-tetraaza-cyclododecane-1,4,10triacetic acid-samarium (III) as a function of pH

The heat treated and untreated samples of Example 41 were each divided into 2 aliquots, the pH of one aliquot adjusted with hydrochloric acid, and the other with sodium hydroxide to the following table. After the samples have reached the desired pH, they are allowed to stand for a further 10 minutes and the metal content of the complexes is determined by the cation exchange method described above. The results are summarized in the following table.

Complex%

PH Hőkczelt Not heat-treated 1 86 83 3 99 99 5 99 99 7 89 96 11 96 89 13 97 97

Example 43 1- (2-Hydroxy-5-nitrobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid-samarium (III) Compound Example 3 χ 10 ² M solution is prepared by dissolving 3.9 mg of compound 260 μΐ ™ Nanopure water. The solution thus obtained was mixed with 10 μΐ 15 microliters of ^2M aqueous 2x10 SmCl3oldattal containing 10 ul of 0.1 N hydrochloric acid as Sm-153 solution (3 x 10 ^-4 mol). The volume is then made up to 9 ml with Dl H2O to 1 ml. The pH was then adjusted to 7 with sodium hydroxide, the solution was split into two 500 μΐ fractions and one fraction was heated at 100 ° C for 1 h. The metal content of the complexes was determined in both heat-treated and non-heat-treated samples. According to the results 71% and 74%, respectively, of the metal content in the heat-treated and non-heat-treated samples.

Example 44 Stabilization of 1- (2-Hydroxy-5-nitrobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid samarium (IIl) as a function of pH

Heat treated and untreated samples of Example 43 were divided into two 250 μΐ aliquots. The pH of one aliquot is adjusted with hydrochloric acid and the other with sodium hydroxide according to the following table. After reaching the desired pH, the samples are allowed to stand for another 10 minutes and the metal content of the complexes is determined according to the method described above. The results are summarized in the following table.

Spreadsheet

PH Complex% heat treated Not heat-treated 1 65 51 3 98 93 5 99 99 7 100 100

HU 219 485 Β

Table (continued)

pH Complex% heat treated Not heat-treated 9 100 99 11 100 96 13 100 98

EXAMPLE 45 1- (2-Hydroxy-5-aminobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid-samarium (III) Compound Example 1 - A ² M solution of (2-hydroxy-5-aminobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid was dissolved by dissolving 3.2 mg in 100 μΐ NANOpure ™ water. 10 µl aliquots of the resulting solution were diluted to 20 μ, and then mixed with 15 μΐ of a ² x 10ól2 M solution of SmClj containing Sm-153. The volume was then made up to ml 1250 μΐ DU H ₂ O and 20 μΐ 0.1N sodium hydroxide. The resulting solution was then split into two 500 μΐ fractions, one of which was heated at 100 ° C for 1 hour. The metal content of the complexes is then determined in both the heat-treated and non-heat-treated samples by the cation exchange method described. If the metal content is less than 95%, the solution is permeated with SP-Sephadex ™ resin and the metal content of the complexes is repeatedly determined. The results are summarized in the following table.

Hőkczclt Not heat-treated Cleaned Not cleaned Cleaned Not cleaned Complex% 99 68 99 21

Example 46 Stabilization of 1- (2-Hydroxy-5-aminobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid samarium (III) as a function of pH

Heat treated and untreated chromatographically purified samples prepared according to Example 45 were divided into two aliquots of 250 μΐ. The pH of one aliquot is adjusted with hydrochloric acid and the other with sodium hydroxide to the pH in the following table. After reaching the desired pH, the samples were allowed to stand for another 10 minutes and the metal content of the complexes was determined by cation exchange. The results are summarized in the following table.

pH Complex% heat treated Not heat-treated 1 60 56 2 74 76

PH Complex% heat treated No heat 3 84 87 5 97 98 7 99 99 9 99 98 11 99 99 13 99 99

Biological examples

Bifunctional chelates containing a metal chelating group such as DTPA and a reactive linker (arylamine) within a molecule are known to be able to covalently bond to various target biomolecules specific for cancer or tumor cell epitopes or antigens. Radioactive nucleus complexes of such conjugates can also be used for diagnostic and / or therapeutic purposes because they are capable of delivering a radioactive nucleus to a cancerous or cancerous cell [Meares et al., Anal. Biochem. 142: 68-78 (1984); see also pp. 215-216. J. Protein Chem., 3 (2), (1984), Meares and Goodwin; and U.S. Patent No. 4,678,677, July 7, 1987; Warshawsky et al., U.S. Patent No. 4,652,519. , March 24, 1987, Brechbiel et al., Inorg Chem., 25, 2772-2781 (1986). It is expected that the product containing the radioactive or luminescent metal may also be used in in vitro immunoassays.

Prior art information

The applicability of labeled antibodies depends on several different factors. For example: 1. the specificity of the antibody, 2. the integrity or stability of the complex under the conditions of use (i.e., serum stability), and 3. the integrity of the antibody, that is, that the specificity and immunoreactivity of the antibody are not affected.

The stability or integrity of the complexes is of particular importance when using the antibody-conjugate containing the radioactive nucleus as diagnostic and / or therapeutic agent. For example, kinetic instability can lead to instability in the serum and cause dissociation of the radioactive nucleus from the complex. As such, it cannot be effectively used for diagnostic and therapeutic purposes. In addition, radiation damage to normal cells is greater [Cole et al., /. Nucl. Med., 28, 83-90 (1987)].

Binding of radioactive nucleus to antibody

The radioactive nucleus is bound to the antibody by either binding the BFC to the antibody in a known manner and then chelating the radioactive nucleus under conditions compatible with the antibody or otherwise binding the antibody to the metal BFC complex under ambient or at elevated temperatures. [Meares et al., Acc. Chem. Res., 17, 202-209 (1984)]. The following examples illustrate this.

HU 219 485 Β

Example ZA (Comparative Example) Binding of 1- (4-Isothiocyanato-benzyl) -diethylene-triamine-pentaacetic acid-samarium-153 complex to IgG or to the F (ab ') ₂ fragment of CC-49;

[ ^{? 53} Sm (SCN-Bz-DTPA)] - IgG and [ ¹⁵³ Sm (SCN-Bz-DTPA)] - F (ab) 2 fragment 1- (4-isothiocyanato-benzyl) -diethylene-triamine-pentaacetic acid- samarium-153 complex [ ¹⁵³ Sm (SCNBz-DTPA)] was prepared by mixing 150 μΐ ¹⁵³ Sm in 0.1 N hydrochloric acid with 9 μΐ 1- (4-isothiocyanato-benzyl) -diethylenetriaminepentaacetic acid HEPES buffer (0.5 mol, pH 8.9, about 30 μΐ) was added to adjust the pH to 6. The resulting ¹⁵³ Sm complex is then mixed with 22.5 χ 10 ~ ⁹ moles of IgG, or CC-49 F (ab ') 2 fragment (1 χ 10 ~ ⁴ moles protein in 50 moles HEPES buffer, pH 8). 5) and adjust the pH to 8.9 with sodium carbonate solution (1 mol, 12-15 μΐ). The conjugation reaction was carried out at room temperature (about 25 ° C) for about 3 hours. The resulting ¹⁵³ Smk complex-labeled IgG or F (ab ') ₂ conjugates were separated, and the XII conjugates were isolated. Example 4 is tested and characterized.

Description of biological experiments

Examples I and 11, Comparative Examples A-D, IN VIVO Test of Bifunctional Chelates The stability of certain rare metal chelates was tested in in vivo animal experiments. For example, Rosoff et al., International Journal of Applied Radiation and Isotopes, 14, 129-135 (1963), disclose the distribution of radioactive rare metal chelates in mice to certain aminocarboxylic acids. It has been found that in vivo competition between chelating agents and various constituents of the body (organic and inorganic) for rare earth ions determines their deposition and excretion. The strong rare earth metal chelates of the present invention exhibit very low dissociation and thus low excretion; whereas the weak and transient chelates known in the art are much easier to associate and thus deposit in various organs such as the liver. However, the concentration of radioactive substances detected in the liver is not always due to poor complexation, but in many cases is due to the affinity between the metal chelate and the liver. Chelates have actually been produced and used to evaluate liver function [Alrit R. Fritzberg, Radiopharmaceuticals: Progress and Clinical Perspectives, Vol. 1, 1986; U.S. Patent Nos. 4,088,747 and 4,091,088.

The bioavailability of the yttrium and samarium chelate of Example 3, which is a typical example of strong rare earth chelates, was examined and the percentage of liver detectable suitable for in vivo qualitative characterization of chelate stability was determined. NTA and EDTA chelates were used for comparison, and samarium chloride in unchelated form was used as a control.

Sprague-Dawley rats (Charles River Laboratories) weighing 150-200 g were placed in cages and provided with unrestricted amounts of drinking water and food. The animals were acclimatized for at least 5 days prior to testing. Prior to injection of the complexes, the animals were placed under a heating lamp (15-30 minutes) to dilate the tail vein. The animals were then placed in the test cage, the tails were cleaned with alcohol swabs and injected via the tail vein (50-200 μΐ). After injection, the animals were transferred to another cage for 2 hours and then sacrificed at the neck. The animals were then autopsied, rinsed with deionized water, dried, weighed and placed in a counter tube. At least three replicates of each material were made and injected. The percentage dose is the number detected in the organ divided by the number detectable in the standard and multiplied by 100 (see table below).

Spreadsheet

Biodegradation data

The example number ligand (example)* Metal injected dose% amount in the liver I 3 Y 0.17 II. 3 sm 0.29 (THE) EDTA sm 8.4 (B) EDTA sm 4.4 (C) NTA sm 8.6 (D) SmCl ₃ sm 39

* The complexes were prepared with a ligand / metal ratio of 1: 1 in the I and II formulations. 5: 1 for example (A) and 300: 1 for examples (B) and (C)

Examples 111 and E)

1: 1 complexes were prepared from Yttrium (containing trace amounts of Y) and Example 3 ligand BA-DOTA and EDTA (Comparative Example E) as described above. 100 μΐ aliquots of the resulting complexes were transferred to separate centrifuge tubes. An excess of Y (III) was then added so that the volume change was minimal and the times recorded. 1.5 hours after the addition of the metal, the percentage of complexes was determined using the cation exchange method described above and compared with the original amount of complexes. The percentage of complexes as a function of the amount of metal added is representative of the lability of the ligand-metal complex. The results are summarized in the following table.

HU 219 485 Β

Spreadsheet

Investigation of complexes

Metal to ligand molar ratio Complex% BA-DOTA EDTA 1 80 98 10 - 86 100 - 78 250 88 48 500 80 16

Example V

a- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium -153 complex;

[ ^I53 Sm (PA-DOTA)] complex

150 μΐ for ¹⁵³ Sm / 0, ln HCl solution (approx

4.6 mCi) 1 μΐ sodium acetate (2 M) and 9 μΐ α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane1.4.7.10-tetraacetic acid ammonium potassium salt (5 mmol in 50 mmol HEPES buffer, pH 8.5, prepared as in Example 9) was added. The resulting mixture was mechanically shaken and then titrated gradually with HEPES buffer (0.5 mol, pH 8.9, about 31 μΐ) to adjust the pH to 7. The resulting mixture was then heated on a sandbath at 98 ° C for 1 hour and then 5 μΐ was used. The assay was performed using Mono-Q ™, HPLC System II, eluting with a gradient solvent system (0-15 min, 0-100% B; A = water and B = 1 molar ammonium acetate and 0.1 mmol EDTA). Yield 85-95% based on ¹⁵³ Sm. The α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7.10-tetraacetic acid, samarium-153 complex thus obtained was compared with the same non-radioactive sample, with suitable chromatographic properties, Mono-Q ™ and GF-250 columns. Further evidence of the presence of the radioactive complex is that it is converted to an isothiocyanate derivative and then bound to an antibody. The complex was prepared even without heating by incubation at ambient temperature for 6-18 hours. The yield in this case was 70-80%.

VI. example

a- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium -153 complex;

[ ^{, 53} Sm (SCN-PA-DOTA)] complex

To the reaction mixture obtained in the previous Example V was added μΐ HEPES buffer (0.5 mol, pH 8.9), 2 μΐ thiophosgene and 0.2 ml chloroform. The resulting mixture is mechanically vigorously shaken two or three times for a few seconds at each shake. The chloroform layer is discarded and the aqueous layer, which predominantly contains the desired product, is further purified. The yield of α- [2- (4-isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium is 85-90%. 153 complexes. ¹⁵³ Sm activity was determined by HPLC using a GF-250 column, HPLC System I. For purification, the aqueous layer was eluted on a Sep-Pak ™ C-18 column with 90% acetonitrile water. The first 300 μΐ was discarded and the next 900 μΐ, which is a derivative of SCN, was used for HPLC analysis. The ^1S3 Sm activity was greater than 90%. The solvent was then evaporated for 1.5-2 hours and the residue used for antibody conjugate preparation.

VII. example

α- (4-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium-153 complex;

[ ^{, 53} Sm (BA-DOTA)] complex

For 200 μΐ of ¹⁵³ Sm / 0.1 l hydrochloric acid, sodium acetate (2 M) and 12 μΐ α- (4-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid (5 mmol in 50 mmol HEPES, pH 8.5, Example 3). The resulting mixture was mechanically stirred and then HEPES buffer (0.5 mol, pH 8.9, approximately 38 μΐ) was added gradually to adjust the pH to 7. The resulting mixture was then heated in a sand bath at 98 ° C for 1 hour. Subsequently, 50 μΐ of the resulting mixture was used for analysis on a mono-Q ™ column (HPLC System II, gradient solvent system: 0-15 min, 0-100% B; A = water, and B = 1 molar). ammonium acetate and 0.1 mmol EDTA). Overall, the yield is 85-95% based on ¹⁵³ Sm. Complex was also prepared by incubation at room temperature for 12-18 hours to give the title compound in 80-90% yield. The title compound thus obtained was also assayed for isothiocyanate derivative on an Mono-Q ™ column and compared to the corresponding sample, and then antibody conjugates were prepared.

Vili. example

α- (4-Isothiocyanato-phenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium-153 complex;

[ ¹³³ Sm (SCN-BA-DOTA) J complex

VII. To the reaction mixture of Example 1 was added 2 μΐ HEPES buffer (0.5 mol, pH 8.9), 2 μΐ thiophosgene and 0.2 ml chloroform. The resulting mixture was mechanically shaken two or three times, each for a few minutes. The chloroform layer was then discarded and the aqueous layer, which predominantly contained the desired product, was further purified. The title compound was obtained in over 90% yield by HPLC analysis on a GF-250 column using HPLC System I. For purification, the aqueous layer was eluted on a Sep-Pak ™ C-18 column with an aqueous solution of 90% acetonitrile. The first 0.3 ml fraction was discarded and the desired fraction was obtained in 86-93% yield (next 1.2 ml fraction). After evaporation of the solvent (ca.

(About 2 hours) was used to prepare the antibody conjugate.

IX. Example 1- 1- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid-samarium-153 complex antibody conjugate [ ^{, 53} Sm (SCN-EA -DO3A)] - IgG conjugate

CC-49 (rat monoclonal IgG), which binds to the TAG-72 epitope (tumor associated antigen), was used as an antibody. Conjugate preparation 187 μΐ antibody solution (1.2 ml of 50 xlO ^-4 mol HEPES buffer, pH 8.5) was mixed with 4.5 mol χ 10 ^{8 153} Sm (SCN-DO3A-EA) complex, which is in Table IV. Prepared as described in Example 1 and then added molar sodium carbonate solution to adjust the pH to 8.9. The reaction proceeded at room temperature for hours and then at ¹⁵³ Sm labeled IgG was separated on Sephadex G-25 (2.2 ml) disposable centrifugal gel filtration column and then further purified by HPLC on GF-250 column using citrate buffer (0.25 M pH 7.4). Fractions containing the labeled IgG were collected, concentrated, and transformed three times into PBS using a Centricon ™ concentrator. The resulting ¹⁵³ Sm-labeled IgG had a specific activity of about 0.16 pCi / pg protein. The intensity of the [ ^{, 53} Sm (EA-DO3A)] - IgG preparation was determined by HPLC [Sivakoff, SI, BioChrom. 1 (1), 42-48 (1986)] and by standard biochemical methods such as sodium dodecyl sulfate: polyacrylamide gel electrophoresis and autoradiography, and solid phase radioimmunoassay (RIA), see David Colcheer et al. Cancer Res. 43: 736-742 (1983).

Example X 1- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid-samarium-153 complex CC-49-F (ab ') _2- fragment conjugate; [ ^I53 Sm (SCN-EA-DO3A)] - F-ab ' ₂ fragment of CC-49

CC-49 F (ab ') ₂ phagem (prepared by the method of Lamoyi and Nisonoff A. by enzymatic digestion), E. Lamoyi and A. Nisonoff, J. Immunol. Methods, 56, 235-243 (1983)] (225 μΐ lxlO- ^4M solution of 50 ml of Hepes buffer, pH 8.5) was mixed with ⁸ moles 2,9xlO ¹⁵³ Sm (SCN-DO3A-EA) complex, which is the ARC. Prepared according to example. To the resulting mixture was then added about 5 pmoles of sodium carbonate to adjust the pH to 8.9 and the reaction was continued for 2.5 hours. The resulting ¹⁵³ Sm fragment is separated and the IX fragment is isolated. Example 4a.

XI. Examples (A and H) Localization in vivo of ⁵³ Sm (EA-DO3A) -IgG and [ ³³³ Sm (EA-DO3A)] F (ab) ₂

The use of ¹⁵³ Sm (EA-DO3A) -labeled IgG (Example IX) and -CC-49 F (ab ') ₂ fragment conjugates (Example X) was characterized by uptake of labeled substances in human tumor-infected athymic mice. Athémyás Female mice (Nu / Nu, CDL-background) subcutaneously (SC) were inoculated (0.1 ml / source) in human colon carcinoma cell line, LS-174 T (about 1 χ 10 ⁶ cells / animal). About 2 weeks after inoculation, each animal was injected via the tail vein with about 2 pCi (15-30 pg) of ¹⁵³ Sm in PBS. The mice were then sacrificed at various time intervals, transfused, and the tumor and selected tissues were excised and weighed and the radioactivity was determined using a gamma counter. The number of strokes per minute (CPM) for ¹⁵³ Sm was determined for each tissue and expressed as CPM per gram tissue per injected dose x 100 (percentage injected dose / g). The results obtained are shown in Figures 1-14. 1A and 1A. and IB. are summarized in tables. For comparison, ¹⁵³ Sm (SCN-BzDTPA) -labelled IgG and -CC-49 F (ab ') ₂ fragment conjugates (Example ZA) were used. The results are shown in the IC and ID tables.

XII. example

α- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-samarium -153 complex [ ¹⁵³ Sm (SCN-PA-DOTA )] - IgG conjugate

The antibody used was CC-49 rat monoclonal IgG, which binds to the epitope of TAG-72 (tumor associated antigen). To prepare the conjugate, 178 μl of antibody solution (1.26 x ^{10 4} moles in 50 mmol HEPES buffer, pH 8.5) was mixed with 3.4 x 10 ^-8 mmol a- [2- (4-isothiocyanato-phenyl) ethyl] -1 With 4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium-153 complex (prepared according to Example VI), then 17 μl of 1 M sodium carbonate solution was added to bring the pH to It is set to 8.9. After 2 hours at room temperature, the ¹⁵³ Sm-labeled IgG was separated by centrifugal gel filtration on a Sephadex ™ G-25 (2.2 mL) disposable column and the resulting material was purified by GF-250 HPLC column elution. citrate buffer (0.25 mol, pH 7.4). Fractions containing the labeled IgG were pooled, concentrated, and exchanged three times with PBS using a Centricum ™ concentrator. The resulting ¹⁵³ Sm-labeled IgG had a specific activity of 0.16 pCi / pg protein. The homogeneity and integrity of the resulting conjugate was confirmed by HPLC and standard biochemical processes (see Example IX).

The following examples illustrate various antibody conjugate production methods, which first include conjugation of BFC to the antibody and then chelation of the BFC-labeled AB conjugate containing the radioactive nucleus.

Example XII A

α- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid - Ab CC ₄₉ Conjugate (IgG CC ₄₉ -PA- DOTA) followed by 26

GB 219 485 ¹⁵³ Β substantially chelating with SM, ¹⁵³ Sm labeled antibody conjugate (IgG _CC-49 -PA ^{DOTA-! S3} preparation Sm) ^{of 153} Sm (PA-DOTA) -jelzésű antibody was prepared so that first BFC such as α- [2- (4-isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid (SCN-PA-DOTA) is added to the antibody at pH 8 And then chelated with ¹⁵³ Sm at pH 6 at room temperature for several hours.

In a typical production method, IgG-CC-49 is concentrated and replaced three times in carbonate buffer (50 mmol, pH 9.1) using a Centricon ™ concentrator (30 K molecular weight cut off) to obtain a solution of antibody concentration> 1.5 to ^{10 4} moles. To make the conjugate, 161 μΐ antibody solution containing 25x10 ^-9 moles of IgG CC-49 was mixed with 5 μΐ SCN-PADOTA (5 mM concentration in the same carbonate buffer prepared as in Example 18). The resulting mixture was allowed to stand at room temperature for 5 hours and then filtered through a 30 K membrane in a Centricon ™ concentrator at 5000 rpm using a Sorvad Rt centrifuge. The resulting antibody conjugate was then washed with 2 mL of 0.25 M DTPA / PBS at pH 7.4, then washed 5 times (2 mL) with MES buffer (20 mmol, pH 5.8), and then for 30 minutes. centrifugation of each fraction. The PA-DOTA-IgG conjugate was then concentrated to a minimum volume (about 100 μΐ) and then checked for integrity by HPLC analysis on a GF-250 column. The conjugate was then purified by mixing 50 μΐ ¹⁵³ Sm / 0.1N hydrochloric acid solution and 20 μΐ MES buffer (1 mol, pH 6) in a vortex mixer and allowed to stand overnight at room temperature. The resulting material was then purified by centrifugal gel filtration and the amount of ¹⁵³ Sm contained in the material was determined by HPLC analysis to be 0.22 BFC-Sm / antibody. The fact that ^I53 Sm ^{binds to} the antibody via chelation with BFC and that this antibody is a true binding covalent and not a non-specific binding was demonstrated by comparison with a control experiment. In the control experiment, IgG-CC-49 solution was mixed with ¹⁵³ Sm mixtures under the same conditions. Similarly, the isolated antibody did not contain detectable levels of ¹⁵³ Sm bound to it. This is evidenced by the non-specific binding between Sm-153 and antibody under these conditions.

XIII. Example a- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-samarium-153 complex - CC-49-F (ab ' ) _2- fragment conjugate; [ ^{, 53} Sm (SCN-PA-DOTA)] - F (ab j ₂ fragment 225 μ-CC-49 F (ab ') ₂ fragment (1 x 10 ^-4 moles in HEPES buffer, pH 8.5) According to E. Lamoyi et al., J. Immunol. Methods, 56, 235-243 (1983), 2.9 x ^{10 8} moles of a- [2- (4-isothiocyanato-phenyl) -ethyl] -1,4,7, With 10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium-153 complex (prepared according to Example VI), about 9 μΐ of 1 M sodium carbonate was added to adjust the pH to 8.9. The reaction is carried out for about 2.5 hours, and the ^<RTIgt ; ^{I53 </RTI>} Sm fragment is isolated and characterized as in Example IX with a specific activity of about 0.4 pCi / pg.

XIV. Examples A and B

IgG and F (ab ') of α- [2- (4-isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium-153 complex ₂ ; In ^{vivo localization of} [ ^1S3 Sm (SCN-PA-DOTA) ^-IgG and [ ^I53 Sm (SCN-PADOTA)] - F (ab ') ₂ Conjugates The title conjugates of XII and

XIII. was used by uptake in athymic mice infected with human tumor. Biolocalization was reported in Section XI. Example IV. The results obtained are shown in Figures 1-7. Figures 8-14 illustrate IgG and Figures 8-14. Figures 1A to 4A for the F (ab ') ₂ fragment and Tables IIA and IIB refer to these results.

For comparison, ^I53 Sm (SCN-Bz-DTPA) IgG and F (ab ') ₂ fragment conjugates (prepared according to Example ZA) were used. The results obtained are shown in Tables IC and ID.

XV. example

α- (4-isothiocyanato-phenyl) -1,4,7,10-tetraaza-cycloaodecane-1,4,7,10-tetraacetic acid-samarium-153 complex antibody conjugate;

[ ¹⁵⁵³ Sm (SCN-BA-DOTA)] - IgG

CC-49 IgG (174 μΐ, 1.2 x 10 ^{-4 -4} moles in 0.25 mole HEPES buffer, pH 8.7) was mixed with 2.0 χ 10 ^-8 moles α- (4-isothiocyanato-phenyl) -1 , 4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid, with samarium-153 complex (2.8 mCi) (prepared according to Example VIII), and then about 2 μΐ of 1 M sodium carbonate solution is added, to keep the pH at 8.7. After 2.5 hours at room temperature, the resulting ^'53 Sm-labeled IgG was separated by centrifugal gel filtration using a Sephadex ™ G-25 (2.2 mL) disposable column followed by HPLC on GF-250. column, eluting with citrate buffer (0.025 mol, pH 7.4). The fractions containing the desired product were combined, concentrated, and exchanged three times with PBS using a Centricon ™ concentrator. The homogeneity and integrity of the samarium-153-labeled IgG preparation was confirmed by HPLC and standard biochemical procedures (see Example IX).

XVI. example α- (4-isothiocyanato-phenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium -153 complex-CC-49-F (ab ') ₂ fragment conjugate; [ ^I53 Sm (SCN-BA-DOTA)] - Fragment CC-49-F (ab ') ₂ CC-49 F (ab') ₂ (84 μΐ 2.4x10 ^-4 mol)

In 0.25 M HEPES buffer, pH 8.7) was prepared by the enzymatic method of Lamoyi et al.

HU 219,485 Β was mixed with 2.1 x 10 ~ ⁸ moles of α- (4-isothiocyanato-phenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium-153 complex (prepared by (Example VIII), then about 2 μΐ of 1 M sodium carbonate solution was added to adjust the pH to 8.7. The reaction was carried out for 2.5 hours, then the resulting ¹⁵³ Sm-labeled fragment was separated and subjected to the reaction of IX. Example 4a.

XVII. Examples (A and B) α- (4-Isothiocyanato-phenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-samarium -153 complex labeled IgG (Example XV) and F (ab ') ₂ fragment (Example XVI);

[ ¹³³ Sm (BA-DOTA)] - In vivo localization of IgG and [ ^l33 Sm (BA-DOTA) jF (ab) ₂

The in vivo test is described in Section XI. Example 14-14. Figures IIIA and IIIA. and IIIB. are summarized in tables.

XVIII. Examples A and B α- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ¹⁷⁷ Lu-complexed IgG and F (ab) _2- fragment;

[ ¹⁷⁷ Lu (PA-DOTA)] - In vivo localization of IgG and j ^I77 Lu (PA-DOTA) jF (ab ') ₂

The preparation and in vivo study of the title compounds are described in V, VI, XI, XII. and XVII. EXAMPLE 1VA and the results obtained are shown in FIG. and IVB. are summarized in tables.

XIX. Examples A and B α- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, Yttrium-90-complexed IgG and F (ab) ^ fragment;

P ° Y (PA-DOTA)] - IgG and [ ^9,> Y (PA-DOTA)] - F (ab) ₂ in vivo localization

In vivo testing of the title compounds is reported in Table XI. except that the tissues were digested and counted by liquid scintillation. The results obtained are reported by VA. and VB. are summarized in tables.

XX. Example ^[33 Sm (BFC) -CC-49 IgG and ^[I77 Lu (BFC) JCC-49 IgG in vitropH stability

[ ¹⁵³ Sm (BFC)] - CC-49-IgG or [ ¹⁷⁷ Lu (BFC) JCC-49-IgG in 0.2 M sodium acetate buffer at pH 6, 4.0 and 2.8, allowed to stand at room temperature with a protein concentration of about 5x10 ⁶ and a concentration of 0.5 complex / antibody. Samples were taken at certain time intervals and analyzed by HPLC (GF-250 column) to determine the degree of lysis of ¹⁵³ Sm or ¹⁷⁷ Lu activity from the protein. Generally, assays were performed for 5 days or until 90% of the radioisotopes were dissociated. The results obtained are expressed relative to the baseline value of the radioisotope or the daily loss value. The results obtained show that ¹⁵³ Sm (PA-DOTA), ⁷⁷ Lu (PA-DOTA), ¹⁵³ Sm (BA-DOTA), i5JSm (PA-DOTMA) and ¹⁵³ Sm (MeO-BA-DOTA) are particularly stable. ) relative to the standard [ ¹⁵³ Sm (Bz-DTPA)] complex at acidic pH.

The results are summarized in the following table.

This greater stability of the complexes in vitro is consistent with the faster clearance of ^{I53 Sm} and ¹⁷⁷ Lu from the body as well as from non-target tissues (e.g., kidney, liver); see pages 1-34. FIGS.

BFC Isotope Isotope% loss / day PH 6.0 4.0 2.8 Bz-DTPA ¹⁵³ Sm <2 35 35 PA-DOTA ¹⁵³ Sm <2 <2 <2 BA-DOTA ¹⁵³ Sm <2 <2 <2 PA-DOTA ¹⁷⁷ Lu <2 <2 <2 MeOBO-DOTA ¹⁵³ Sm <2 <2 <2 EA DO3A ¹⁵³ Sm <2 90 95 PA-DOTMA ¹⁵³ Sm <2 <2 -

XXI. Examples (A and B) are α- (2-Methoxy-5-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid samarium-153 complex-labeled IgG and F (ab ') ₂ fragment conjugates; ^{Localization of} [ ^I53 Sm (MeO-HA-DOTA)] - IgG and [ ^15S Sm (MeO-BA-DOTA)] - F (ab) ₂

The preparation of the title compounds is described in V, VI, XII. and XIII. and in vivo tests were performed as described in Example XI. Example 3a. The results obtained are provided by VIA. and VIB. are summarized in tables.

XXII. Example ¹³³ Preparation of a Sm (PA-DOTMA) complex

100 microliters of ¹⁵³ Sm solution (0.3 x 10 ~ ³ moles in 0.01 N HCl, 5 mCi) were mixed with 6 μΐ PA-DOTMA (diastereomer with higher R R value in Example 29, 5 mmol Milli-Q). ™ in water) and 20 μΐ MES buffer (1 mol, pH 6) and 1 μΐ HEPES buffer (0.5 mol, pH 8.7). The resulting solution was stirred in a vortex mixer to give a final pH of 6. The resulting mixture was then heated at 90 ° C for 30 minutes and analyzed by HPLC (GF-250 column) to determine the degree of complexation. The result obtained is 90% or more. The resulting ¹⁵³ Sm-PA-DOTMA complexes were compared with the corresponding non-radioactive Sm complex, prepared as described in Example 42 and tested.

XXIII. Example ^53, Preparation ^{of 53} Sm (SCN-PA-DOTMA)

In the XXII. The ¹⁵³ SmPA-DOTMA complex prepared in Example 1 is kept at room temperature for 20 minutes and then 10 μΐ of 1% thiophosgene and 90%

EN 219 485 Β mixture containing acetonitrile / water. The resulting mixture was vigorously stirred in a Vortex mixer and allowed to stand at room temperature for 5-10 minutes. The reaction is spontaneous according to HPLC analysis. The activated purified ¹⁵³ Sm complex is extracted with 3 x 200-200 μΐ chloroform and the aqueous layer is pre-treated with 5 ml of PRP packed (PRP packed with: Miniclean ™ pack, Alltech Associates, Deerfield, IL). methanol and 5 mL of MES buffer (20 mmol, pH 5.8). After the column was washed with 5 mL of 10 volume MES buffer was passed through 5 ml of Milli-Q ™ water and extracted with 900 μΐ 90% acetonitrile in the resulting material to give 90% strength Smaktivitást ^153, the first 100 μΐ fraction was discarded.

The mixture thus obtained is evaporated to dryness and concentrated under reduced pressure at a temperature below 40 ° C.

XXIV. Examples A and B Conjugation of ⁵³ Sm (SCN-PA-DOTMA) to IgG and CC-49 F (ab ') ₂ Generally, the antibody is concentrated and ion-exchanged with carbonate buffer (pH = 9.1 or 9.5, 5 mmol) in a Centricon ™ concentrator (30 K cut-off molecular weight) to achieve a protein concentration of 1.5 χ 10 ^-4 molar or greater.

Conjugate preparation of small quantities of carbonate, which is 1.5 χ 10 to provide 30 concentration ^{of 4M} final protein, mixed with ¹⁵³ Sm (PADOTMA) izotiocianátoszármazékával (prepared as described in Example XXIII.) And the mixture concentrated antibody was added to the BFC - ¹⁵³ Sm complexes in equimolar amounts. The mixture was stirred in a vortex mixer and allowed to stand at room temperature for 1 hour. During this time, 40-50% of the ¹⁵³ Sm bound to the antibody was detected by HPLC analysis on a GF-250 column. The labeled antibody was then isolated by two successive centrifugal gel filtration on Sephadex ™ G-250 columns (2.2 mL). The homogeneity, integrity, and immunoreactivity of the labeled antibody were assayed by HPLC analysis and standard biochemical methods as described above.

XXV. Examples A and B ^{Bioassay of I53} Sm (PA-DOTMA) -labeled IgG and CC-49 F (ab) ₂ Example IV. The results obtained are reported in VIIA. and VIIB. and Tables 15-28. Figs.

Spreadsheet

conjugates Signal Spreadsheet Figures Biological example [ ¹⁵³ Sm (EA-DO3A)] - IgG-CC-49 Δ IA. 1-7. IX. [ ¹⁵³ Sm (EA-DO3A)] - F (ab ') ₂ -CC-49 Δ IB. 8-14. X.

XXVI. Example ⁷⁷ Lu (PA-DOTMA) complex μΐ mixed solution of ¹⁷⁷ Lu-O ⁽³ mol 6xl0- 1.1 N HCl benzodiazepine 4 mCi) PA-DOTMA μΐ 36 (prepared as in Example 29), and a 5 mmol solution in milli-Q ™ water. This solution was mixed in a vortex mixer and 115 μΐ MES buffer (1 mol, pH 6.0) was added to adjust the pH of the solution to 5.5-6. The resulting solution was stirred at 90 ° C for 30 minutes, whereby a chelation greater than 90% was observed. The mixture was then passed through a column pre-treated with 5 mL of PRP filled with 5 mL of methanol and 5 mL of MES buffer (20 mmol, pH 5.8), washed with 5 mL of milli-Q ™ water, extracted with 1000 μΐ 90% acetonitrile 15 and determine the ¹⁷⁷ Lu activity, which is 78% of the baseline value (not counting the first 100 μΐ extract discarded).

XXVU. EXAMPLE ^I77 Lu (SCN-PA-DOTMA)

In the XXVI. The 90% acetonitrile ¹⁷⁷ Lu (PA-DOTMA) complex solution prepared in Example 1 is mixed with 6 μΐ of a 90% acetonitrile solution containing 10% thiophosgene. The resulting solution was stirred in a vortex mixer, and after 25 minutes, the amount of isothiocyanate derivative was determined by HPLC. The reaction is generally quantitative. The reaction mixture was then stripped of solution and excess thiophosgene by evaporation under reduced pressure at 40 ° C for 1-2 hours. The residue is used for conjugation with antibody.

XXVIII. Example ¹⁷⁷ Conjugation of Lu (PA-DOTMA) with IgG-CC-49

The IgG-CC-49 antibody was mixed in carbonate buffer (50 mmol, pH 9.1) in an equimolar amount of XXVII. with the isothiocyanate derivative prepared according to example. The reaction is carried out for 70 minutes at a antibody concentration of 1.5 χ 10 ^-4 mole, and the labeled antibody is isolated and then isolated in IX. Example 4a.

XXIX. Example ^I77 Lu (PA-DOTMA) and ⁷⁷⁷ Lu (PA-DOTA) Long term testing of 45 CC-49 IgG labeled biodistribution

The long-term study in animals was performed with ¹⁷⁷ Lu-labeled antibodies taking into account the long half-life of this material (161 hours). The assay was performed on Balb / c mice for a period of 33 weeks, as described in Table XI. Example 50. The results obtained are shown in Figure VIIIA. and Table 29-34. Figs.

The following notations are used in the figures and in the following tables.

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Table (continued)

conjugates Signal Spreadsheet Figures Biological example [ ¹⁵³ Sm (Bz-DTPA)] - IgG-CC-49 0 IC. 1-7. ZA [ ¹⁵³ Sm (Bz-DTPA)] - F (ab ') ₂ -CC-49 She ID. 8-14. ZA [ ¹⁵³ Sm (PA-DOTA)] - IgG-CC-49 □ IF. 1-7. XII. [ ¹⁵³ Sm (PA-DOTA)] - F (ab ') ₂ -CC-49 □ IIB. 8-14. XIII. [ ¹⁵³ Sm (BA-DOTA)] - IgG-CC-49 IIIA. 1-7. XV. [ ¹⁵³ Sm (BA-DOTA)] - F (ab ') ₂ -CC-49 IIIB. 8-14. XVI. [ ¹⁵⁵³ Sm (PA-DOTMA)] - IgG-CC49 V VIIA. 15-21. XXIV. [ ¹⁵³ Sm (PA-DOTMA)] - F (ab ') ₂ -CC-49 V VIIB. 22-28. XXV. [ ¹⁷⁷ Lu (PA-DOTMA)] - IgG-CC49 • VIIIA. 29-34. XXIX. [ ^l77 Lu (PADED)] - IgG-CC-49 ► VIIIA. 29-34. XXIX.

In the figures attached to the description, the data are as follows:

Figure Organ Title 25 First Blood ¹⁵³ Bio-distribution of Sm-BFC * Second Liver ¹⁵³ Bio-distribution of Sm-BFC * Third spleen ¹⁵³ Bio-distribution of Sm-BFC * 4th Kidney ¹⁵³ Bio-distribution of Sm-BFC * 30 5th Tumor ^l53 Sm-BFC * biodistribution 6th thigh bone ¹⁵³ Bio-distribution of Sm-BFC * 7th All testretenció ¹⁵³ Bio-distribution of Sm-BFC * 8th Blood ¹⁵³ Bio-distribution of Sm-BFC ** 35 9th Liver ¹⁵³ Bio-distribution of Sm-BFC ** 10th spleen ¹⁵³ Bio-distribution of Sm-BFC ** 11th Kidney ¹⁵³ Bio-distribution of Sm-BFC ** 12th Tumor ¹⁵³ Bio-distribution of Sm-BFC ** 13th thigh bone ¹⁵³ Bio-distribution of Sm-BFC ** 40 14th All testretenció ¹⁵³ Bio-distribution of Sm-BFC ** 15th Blood ¹⁵³ Bio-distribution of Sm-BFC * 16th Liver ^l53 Sm-BFC * biodistribution 45 17th spleen ¹⁵³ Bio-distribution of Sm-BFC * 18th Kidney ¹⁵³ Bio-distribution of Sm-BFC * 19th Tumor ¹⁵³ Bio-distribution of Sm-BFC * 20th thigh bone ¹⁵³ Bio-distribution of Sm-BFC * 21st All testretenció ¹⁵³ Bio-distribution of Sm-BFC * 50

Figure Organ Title 22nd Blood ^I53 Sm-BFC ** biodistribution 23rd Liver ¹⁵³ Bio-distribution of Sm-BFC ** 24th spleen ¹⁵³ Bio-distribution of Sm-BFC ** 25th Kidney ^I53 Sm-BFC ** biodistribution 26th Tumor ¹⁵³ Bio-distribution of Sm-BFC ** 27th thigh bone ¹⁵³ Bio-distribution of Sm-BFC ** 28th All testretenció ¹⁵³ Bio-distribution of Sm-BFC ** 29th Blood ¹⁵³ Bio-distribution of Sm-BFC *** 30th Liver ¹⁵³ Bio-distribution of Sm-BFC *** 31st spleen ¹⁵³ Bio-distribution of Sm-BFC *** 32nd Kidney ¹⁵³ Bio-distribution of Sm-BFC *** 33rd Tumor ¹⁵³ Bio-distribution of Sm-BFC *** 34th thigh bone All ¹⁵³ Bio-distribution of Sm-BFC *** testretenció ¹⁵³ Bio-distribution of Sm-BFC ***

* In a naked mouse with CC ₄₉ -IgG LS174-T tumor.

** In a naked mouse with CC ₄₀ -F (ab ') ₂ LS174-T tumor. *** CC ₄₉ -lgG in Balb / c mouse.

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IA. [ ¹⁵³ Sm (EA-DO3A)] - Bioavailability of ¹⁵³ Sm injected as IgG-CC-49, injection dose / gram% (n = 5)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 23.08 2.36 17.25 2.40 11.32 * 1.06 5.78 * 1.69 Liver 6.95 1.28 6.58 0.56 6.58 0.62 7.62 0.98 spleen 5.17 0.59 5.06 0.96 4.62 0.72 4.92 1.72 Kidney 4.59 0.68 4.01 0.53 3.74 0.76 3.20 0.59 Tumor 11.44 4.13 27.19 2.12 53.01 9.10 59.01 13.23 thigh bone - - 2.58 0.52 2.78 0.20 4.22 0.17 Tumor weight / g 0.16 0.09 0.20 0.09 0.30 0.17 0.52 0.34

* n = 4.

IB. [ ¹⁵³ Sm (EA-DO3A)] - Bioavailability of ¹⁵³ Sm injected in F (ab ') ₂ -CC-49 injection dose / g% (n = 5)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 13.36 0.97 1.52 0.42 0.20 0.06 0.05 0.01 Liver 7.13 0.65 8.59 1.3 8.08 0.8 8.11 0.92 spleen 5.94 1.24 4.96 1.27 4.81 0.99 5.08 1.43 Kidney 41.60 5.60 64.32 7.88 64.12 12.59 * 37.63 6.57 Tumor 12.95 1.73 16.84 3.71 11.50 4.56 5.53 1.46 thigh bone 2.74 0.65 2.45 0.26 3.41 0.75 4.99 0.29 Tumor weight / g 0.25 0.20 0.34 0.25 0.28 0.14 0.44 0.27

* n = 4.

IC. [ ¹⁵³ Sm (Bz-DTPA)] - ^{Bio-distribution of I53} Sm injected as IgG-CC-49, injection dose / gram% (n = 10)

Organ clock clock clock

120 hours

AVG STD AVG STD AVG STD AVG STD Blood 24.97 3.07 16.05 2.45 12.81 2.69 5.84 1.76 Liver 7.74 1.35 6.01 0.95 5.68 1.07 5.43 1.45 spleen 5.69 1.04 4.86 1.36 4.72 0.81 3.78 0.63 Kidney 4.52 1.04 3.96 0.88 3.81 0.61 3.76 0.31 Tumor 13.90 3.18 42.86 14.03 46.33 8.30 61.32 19.80 thigh bone 2.36 0.12 1.95 0.38 1.97 0.75 2.25 0.50 Total body retention 85.45 4.74 80.16 3.58 77.36 5.32 70.60 3.32

ID. [ ¹⁵³ Sm (Bz-DTPA)] - ^{Bio distribution of I53} Sm injected as F (ab ') ₂ -CC-49, injection dose / gram% (n = 10)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 15.03 1.84 2.13 0.48 0.29 0.08 0.04 0.02 Liver 7.07 0.88 7.03 1.24 6.14 0.67 5.80 1.00 spleen 4.50 0.97 4.22 1.15 3.65 0.62 3.24 0.42 Kidney 46.74 8.69 80 12.76 61.42 9.85 30.17 5.17

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ID. Table (continued)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Tumor 15.95 3.88 18.66 5.10 15.55 2.80 6.54 1.14 thigh bone 2.48 0.24 1.77 0.28 2.48 0.40 2.85 0.35 Total body retention 83.23 4.64 72.17 6.20 58.89 4.40 40.53 2.48

IIA. [ ¹⁵³ Sm (PA-DOTA)] - Bio distribution of ¹⁵³ Sm injected as IgG-CC-49, injection dose / gram% (n = 5)

Organ 5.5 hours 25 hours 49 hours 121 hours AVG STD AVG STD AVG STD AVG STD Blood 24.65 2.89 16.18 2.10 13.22 1.08 7.34 4.23 Liver 8.25 1.14 5.92 0.41 5.59 0.52 4.16 1.03 spleen 6.98 1.70 5.05 0.20 4.27 0.52 4.31 L95 Kidney 4.01 0.86 3.16 0.55 3.44 0.39 3.26 0.57 Tumor 14.31 * 2.00 42.81 8.83 72.59 21.53 78.36 33.29 thigh bone 2.69 0.41 2.02 0.32 1.69 0.37 1.44 0.69 Tumor weight / g 0.27 0.29 0.34 0.26 0.16 0.13 0.28 0.15

* N = 4th

IIB. [ ¹⁵³ Sm (PA-DOTA)] - Bio distribution of ¹⁵³ Sm injected as F (ab ') ₂ -CC-49, injection dose / gram% (n = 5)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 14.74 0.76 2.25 0.45 0.33 0.08 0.02 0.01 * Liver 7.83 0.82 5.04 0.32 * 4.48 0.41 1.61 0.26 spleen 4.80 0.84 3.27 0.45 3.33 0.82 1.41 0.10 Kidney 51.28 5.83 78.78 8.40 61.91 8.17 19.79 5.75 Tumor 17.99 6.53 22.09 6.07 14.93 1.91 5.81 1.21 thigh bone 2.43 0.17 1.34 0.25 1.17 0.22 0.51 0.18 Tumor weight / g 0.20 0.08 0.19 0.13 0.29 0.10 0.45 0.23

* N = 4th

IIIA. Table ¹⁵³ [ ¹⁵³ Sm (BA-DOTA)] - Biodistribution of ¹⁵³ Sm injected as IgG-CC-49, injection dose / gram% (n = 5)

Organ 5.5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 24.89 1.29 17.58 1.79 13.61 0.53 8.68 2.94 Liver 7.53 0.79 5.46 * 0.62 5.35 * 0.40 4.26 * 0.58 spleen 5.52 0.61 5.83 1.07 4.78 0.35 3.76 1.28 Kidney 3.84 0.50 3.96 * 0.36 3.17 * 0.57 3.49 * 0.43 Tumor 14.41 2.96 38.65 6.45 55.84 11.43 85.26 25.70 thigh bone 2.54 0.34 1.81 0.35 2.08 0.30 L17 0.48 Tumor weight / g 0.34 0.22 0.13 0.04 0.20 0.06 0.20 0.15

* N = 4th

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ΙΠΒ. [ ¹⁵³ Sm (BA-DOTA)] - Bioavailability of ⁵³ Sm injected in F (ab ') ₂ -CC-49 injection dose / g% (n = 5)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 15.62 * 0.86 2.42 0.42 0.28 0.08 0.03 0.01 Liver 7.69 0.71 6.18 0.82 4.22 0.88 1.56 0.25 spleen 4.93 0.63 3.94 0.52 2.88 * 0.36 1.43 * 0.30 Kidney 50.29 4.99 77.19 * 3.22 60.44 7.71 24.98 2.67 Tumor 14.58 2.24 24.28 1.81 17.65 3.25 7.39 2.54 thigh bone 2.13 0.18 1.36 0.15 1.03 0.28 0.68 0.35 Tumor weight / g 0.17 0.02 0.12 0.07 0.17 0.06 0.12 0.10

* N = 4th

IVA. [ ¹⁷⁷ Lu (PA-DOTA)] - Bio-distribution of ¹⁷⁷ Lu injected as IgG-CC-49, injection dose / gram% (n = 5)

Organ 5 hours 24 hours 48 hours 121 hours AVG STD AVG STD AVG STD AVG STD Blood 26,50 4.32 19.43 * * 1.59 14.89 1.72 11.73 3.14 Liver 7.47 1.34 * 6.74 * 0.31 5.11 0.41 4.71 0.63 spleen 6.29 1.56 * 5.47 * 1.24 4.43 0.31 5.35 1.82 Kidney 3.86 * 0.80 * 4.13 * 0.45 3.13 * 0.30 3.57 1.15 Tumor 11.94 3.39 49.03 * * 3.21 56.36 * 4.24 92.05 15.32 * thigh bone 2.79 1.08 * 2.15 * 0.30 2.11 0.39 1.32 * 0.19 Tumor weight / g 0.13 0.09 * 0.09 0.02 * 0.08 0.02 * 0.10 * 0.04

* N = 4th

IVB. [ ¹⁷⁷ Lu (PA-DOTA)] - Bio-distribution of ¹⁷⁷ Lu injected in F (ab ') ₂ -CC-49 injection dose / g% (n = 5)

Organ 5 hours 24 hours 48 hours 121 hours AVG STD AVG STD AVG STD AVG STD Blood 12.65 * 0.87 * 2.40 * 0.18 0.47 0.08 0.04 0.02 * Liver 5.39 * 0.27 * 5.54 * 0.31 3.25 0.45 1.30 * 0.24 spleen 4.21 0.95 * 3.00 * 0.55 2.60 0.74 1.33 0.27 Kidney 38.82 6.42 74.40 * * 6.13 48.45 6.47 15.88 2.13 Tumor 10.79 2.69 19.10 * * 2.08 15.44 2.35 4.69 0.78 thigh bone 2.13 0.83 * 1.45 * 0.33 0.95 0.43 0.41 0.25 * Tumor weight / g 0.10 0.03 * 0.10 * 0.03 0.07 0.06 0.19 0.13

* N = 4th

VA. Table 9 [9 ° Y (PA-DOTA)] - Biodistribution *> Υ of IgG-CC-49 injected dose / g%

Organ n = 3 5 hours n = 5 24 hours n = 5 48 hours n = 5 120 hours AVG STD AVG STD AVG STD AVG STD Blood 25.15 1.96 18.22 2.69 15.85 * 0.81 6.62 2.94 Liver 8.25 0.42 7.61 0.77 6.52 0.96 5.68 1.26 spleen 5.13 0.26 5.50 0.72 5.66 * 0.38 4.19 0.88

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VA. Table (continued)

Organ n = 3 5 hours n = 5 24 hours n = 5 48 hours n = 5 120 hours AVG STD AVG STD AVG STD AVG STD Kidney 3.04 0.58 4.35 1.06 3.66 0.60 2.47 0.33 Tumor 12.45 0.79 44.73 ** 1.43 68.41 * 5.48 70.32 21.34 thigh bone 2.05 0.15 1.81 0.54 1.79 * 0.11 1.04 0.45

* N = 4th

** n = 3rd

VB. Table ⁹⁰ [ ⁹⁰ Y (PA-DOTA)] Bio-distribution of ⁹⁰ Y injected in F (ab ') ₂ -CC-49 injection dose / g% (n = 5)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 13.87 1.19 2.22 0.46 0.31 * 0.03 0.04 0.02 Liver 6.98 0.84 4.13 * 0.42 3.01 0.33 1.56 0.32 spleen 4.27 0.35 3.44 * 0.49 2.54 0.71 1.33 0.54 Kidney 44.60 5.52 65.40 5.92 42.31 4.67 12.36 2.60 Tumor 13.08 * 0.88 22.44 4.77 17.84 5.09 5.83 2.09 thigh bone 1.42 0.22 1.12 0.14 0.39 0.18 0.38 0.12

* N = 4th

VIA. [ ¹⁵³ Sm (MeO-BA-DOTA)] - Biodistribution of ¹⁵³ Sm injected as IgG-CC-49, injection dose / gram%

Organ n = 5 5 hours n = 5 24 hours n = 5 48 hours n-4 120 hours AVG STD AVG STD AVG STD AVG STD Blood 21.06 * 0.75 14.57 1.18 13.98 0.78 9.49 1.06 Liver 9.00 1.93 6.22 0.68 6.32 0.41 4.34 0.27 spleen 5.39 1.40 4.00 0.64 4.69 0.93 4.53 0.41 Kidney 3.48 * 0.24 3.23 0.41 3.47 0.45 3.47 0.19 Tumor 18,00 8.91 41.64 3.37 58.34 5.78 69.26 21.92 thigh bone 2.54 0.27 1.94 0.32 2.05 0.29 1.29 0.16

* N = 4th

VIB. [ ¹⁵³ Sm (MeO-BA-DOTA)] - Biodistribution of ¹⁵³ Sm injected as IgG-CC-49, injection dose / gram%

Organ n = 5 5 hours n = 5 24 hours n = 4 48 hours n = 4 120 hours AVG STD AVG STD AVG STD AVG STD Blood 12.98 ** 0.36 2.13 0.37 0.31 * 0.03 0.05 0.03 Liver 6.58 ** 0.16 6.77 1.13 4.62 0.27 2.46 0.55 spleen 3.95 0.57 3.53 0.76 2.59 0.40 2.18 0.68 Kidney 47,07 5.85 74,16 8.03 47.68 6.29 24.25 2.74 Tumor 12.59 3.96 17,30 1.68 12.33 * 0.80 5.21 * 0.63 thigh bone 2.61 0.30 1.61 0.31 0.90 0.03 0.68 ** 0.10

* N = 3rd

** n = 4th

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VUA. [ ¹⁵³ Sm (PA-DOTMA)] - Bioavailability of ¹⁵³ Sm injected as IgG-CC-49, injection dose / gram% (n = 5)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 23.00 1.11 15.58 1.37 11.78 0.36 8.98 0.91 Liver 7.71 0.51 6.68 1.16 4.98 0.32 4.72 0.69 spleen 6.05 1.03 5.28 0.45 3.66 0.35 3.72 0.53 Kidney 3.68 * 0.23 4.03 0.41 3.95 0.15 * 3.17 0.86 Tumor 10.40 2.10 31.85 * 2.52 44.70 10.03 66.45 13.60 thigh bone 1.94 0.20 1.66 0.11 1.49 0.10 1.22 0.08

* N = 4th

V11B. [ ¹⁵³ Sm (PA-DOTMA)] - Bioavailability of ¹⁵³ Sm injected as F (ab ') ₂ -CC-49, injection dose / g% (n = 5)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 12.85 1.06 1 0.13 0.38 0.09 0.07 0.02 Liver 6.12 0.52 5.36 0.49 3.65 0.65 1.93 0.32 spleen 4.08 0.37 2.88 0.31 2.09 0.62 0.92 * 0.13 Kidney 50.28 5.26 58.59 5.63 45.29 7.04 14.85 * 0.66 Tumor 11.85 2.23 14.27 1.02 15.26 2.83 4.90 2.01 thigh bone 1.77 0.06 1.06 0.13 0.96 0.28 0.79 0.24

* N = 4th

VIIIA. [ ¹⁷⁷ Lu (PA-DOTMA)] - Bio-distribution of ¹⁷⁷ Lu injected as IgG-CC-49, injection dose / gram% (n = 5)

Organ 24 hours 48 hours 7 days 14 days 21 days AVG STD AVG STD AVG STD AVG STD AVG STD Blood 31.35 2.66 30.25 * 0.93 22.47 0.71 14,13 1.05 9.88 0.83 Liver 10.01 0.46 11.22 1.47 8.61 0.79 6.76 0.67 5.41 1.39 spleen 10.13 1.16 10.91 * 0.67 10.79 * 0.36 8.09 0.98 6.03 0.55 Kidney 6.52 2.06 6.14 0.67 5.21 0.97 3.44 0.43 2.17 0.22 thigh bone 3.97 0.45 4.53 0.38 2.84 0.16 2.36 0.23 1.67 0.30

* N = 4th

VII1B. [ ¹⁷⁷ Lu (PA-DOTA)] - Bio-distribution of ¹⁷⁷ Lu injected as IgG-CC-49, injection dose / gram% (n = 5)

Organ 24 hours 48 hours 7 days 14 days 21 days AVG STD AVG STD AVG STD AVG STD AVG STD Blood 30.23 * 0.57 28.42 2.02 21.96 1.03 14,73 0.48 8.85 1.03 Liver 11.54 1.34 10.84 1.27 9.20 1.04 6.69 0.48 6.07 1.41 spleen 10.86 1.47 11.55 1.64 10.62 1.25 9.25 1.10 6.64 1.43 Kidney 7.67 1.83 5.80 0.59 4.98 0.50 3.27 * 0.19 2.60 0.64 thigh bone 3.65 0.62 3.64 0.51 2.98 0.41 2.50 0.20 1.17 * 0.17

* N = 4th

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Embodiments other than the preceding examples, which will be apparent to those skilled in the art, are also within the scope of the invention. The examples provided are merely illustrative of the invention and are not intended to limit the claims in any way.

Claims (61)

PATENT CLAIMS A process for the preparation of compounds of formula IA and pharmaceutically acceptable salts thereof wherein Q is independently hydrogen or (CHR ⁵ ) _p CO ₂ R; Q ¹ is hydrogen or -CO ₂ R, in which R is hydrogen or (C 1 -C 4) -alkyl, provided that Q and Q ¹ together are not at least two hydrogen, and when Q ¹ is hydrogen, R ³ must be other than hydrogen, R ⁵ is hydrogen or C 1-4 alkyl, n is a number from 0 to 5, p is 1 or 2, R ² is hydrogen, nitro, amino or isothiocyanate; R ³ is C 1-4 alkoxy, hydroxy, or hydrogen, R ⁴ is hydrogen, nitro, amino or isothiocyanate, provided that R ² and R ^{4 are} not simultaneously hydrogen, but one of R ² and R ⁴ must be hydrogen, characterized in that a) reacting a compound of formula IA 'wherein Q ¹ , R ² , R ³ , R ⁴ and n are as defined above, with a compound capable of introducing a - (CHR ⁵ ) _p CO ₂ R group, or b) for the preparation of compounds of formula IA in which R ² or R ⁴ is amino and the other substituents are represented by a compound of formula (LA) wherein R ² or R ⁴ is nitro, or c) for the preparation of a compound of formula IA wherein R is hydrogen and the other substituents are hydrolyzed according to the present invention to a compound of formula IA having one of R 1 -C 4 alkyl; or preparing d) (IA) wherein R ² or R ⁴ isocyanato group and the other substituents are thiophosgene stated above, with an R ² or a compound of formula (LA) ⁴ is an amino group R is reacted wherein, and any resulting optionally converting the compound into a pharmaceutically acceptable salt. 2. A process according to claim 1 for the preparation of the pharmaceutically acceptable salts of the compounds of formula (IA) with potassium, sodium, lithium, ammonium, calcium, magnesium or hydrochloride salts by reacting the corresponding starting compounds. and converting the resulting compounds of formula IA into salts. 3. A method according to claim 1 for the preparation of (IA) compounds of the formula wherein R and R ⁵ each represent a hydrogen atom, the other substituents are as defined in claim 1, characterized in that using the corresponding starting materials. A process for the preparation of a compound of formula IA according to claim 1, wherein R is hydrogen, R ⁵ is methyl, and the other substituents are as defined in claim 1, wherein the appropriate starting materials are used. A process for the preparation of a compound of formula IA according to claim 1, wherein R ³ and R ² are hydrogen, the other substituents according to claim 1, characterized in that the appropriate starting materials are used. 6. A process according to claim 1 for the preparation of a compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein: Q is hydrogen or -CHR ⁵ CO ₂ R, R is hydrogen or C 1 -C 4 alkyl, provided that at least two of Q are other than hydrogen and R ³ is other than hydrogen, n is 0-5 number, and Of R ^2, R ⁴ and R ⁵ are as defined in claim 1, characterized in that using the corresponding starting materials. 7. A process according to claim 1 for the preparation of compounds of formula (IV) and pharmaceutically acceptable salts thereof, wherein: Q and n are as defined in claim 1, with the proviso that at least two of Q are other than hydrogen, R ³ is C 1-4 alkoxy or hydroxy, R ⁴ is nitro, amino or isothiocyanate, characterized in that the appropriate starting materials are used. The process for preparing 1- (2-methoxy-5-aminobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid according to claim 7, characterized in that the corresponding starting material is materials. The process according to claim 7 for the preparation of 1- (2-hydroxy-5-aminobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid, characterized in that materials. 10. A process according to claim 1 for the preparation of compounds of formula (VI) and pharmaceutically acceptable salts thereof, wherein: HU 219 485 Β Q is hydrogen or CHR ⁵ CO ₂ R, R is hydrogen or C 1 -C 4 alkyl, provided that at least one of Q is other than hydrogen, n is a number from 0 to 5, R ² , R ³ , R ⁴ and R ⁵ according to claim 1, characterized in that the appropriate starting materials are used. 11. A process for the preparation of a compound of formula VII according to claim 10, or a pharmaceutically acceptable salt thereof, wherein R, Q and n are as defined in claim 10, R ² is nitro, amino or isothiocyanate, characterized in that: using the appropriate starting materials. 12. All. The process according to claim 1, which is la- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1- (R, S) -acetic acid-4,7,10-tris- [ (R) -α-methyl-acetic acid] -1-isopropyl-4,7,10-trimethyl ester, characterized in that the appropriate starting materials are used. 13. The process according to claim 11, wherein lα- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1 (R, S) -acetic acid-4,7,10- Tris - [(R) -α-methyl-acetic acid], characterized in that the appropriate starting materials are used. 14. The process according to claim 11, wherein lα- [2- (4-isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraaza-cyclododecane-1- (R, S) -acetic acid-4,7,10. -tris - [(R) -α-methyl-acetic acid], characterized in that the appropriate starting materials are used. The process according to claim 11 for the preparation of a mixed ammonium sodium salt of α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetic acid , characterized in that the appropriate starting materials are used and the salt-conversion step is carried out. The process according to claim 11 for the preparation of α- (4-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetic acid, characterized in that the appropriate starting materials are used. 17. A process according to claim 11 for the preparation of α- (4-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,10-triacetic acid, wherein the appropriate starting materials are used. 18. A process according to claim 11 for the preparation of α- (4-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid, wherein the appropriate starting materials are used. 19. A process according to claim 11 for the preparation of α- (4-isothiocyanato-phenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid, characterized in that the appropriate starting materials are used. 20. The process of claim 11 is α- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-1,4,7, 10-tetramethyl ester, characterized in that the appropriate starting materials are used. The process according to claim 11, which is α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid-1,4,7 10-tetramethyl ester, characterized in that the appropriate starting materials are used. 22. A process according to claim 11 for the preparation of α- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid, characterized in that suitable starting materials are used. A process according to claim 11 for the preparation of α- [2- (4-isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4.7.10-tetra-acetic acid, wherein starting materials are used. 24. A process according to claim 1 for the preparation of compounds of formula (VIII) and pharmaceutically acceptable salts thereof, wherein: Q is hydrogen or -CHR ⁵ CO ₂ R, R and ^R5 are hydrogen or C1 -C4 alkyl, with the proviso that at least one Q is a hydrogen atom, n is a number from 0-5. R ³ is as defined in claim 1 and R ⁴ is nitro, amino or isothiocyanate - characterized in that the appropriate starting materials are used. 25. A process according to claim 24 for the preparation of α- (2-methoxy-5-nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid, characterized in that the appropriate starting materials are used. . 26. A process according to claim 24 for the preparation of the tetraammonium salt of α- (2-methoxy-5-nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid, characterized in that materials and carrying out the salt conversion step. 27. A process according to claim 24 for the preparation of a tetraammonium salt of α- (2-methoxy-5-isothiocyanato-phenyl) -1,4,7,10-tetraaza-cyclododecane-1,4.7.10-tetraacetic acid, characterized in that the appropriate starting materials are used. , and performing the salt conversion step. A process for use as a ligand of a compound of formula IA or a pharmaceutically acceptable salt thereof Q is independently hydrogen or - (CHR ⁵ ) _p CO ₂ R, Q ¹ is hydrogen or -CO ₂ R is in which R is hydrogen or (C 1 -C 4) -alkyl, provided that Q and Q ¹ together are not at least two hydrogen, and when Q ¹ is hydrogen, R ³ must be other than hydrogen, R ⁵ is hydrogen or C 1-4 alkyl, n is a number from 0 to 5, p is 1 or 2, R ² is hydrogen, nitro, amino or isothiocyanate; HU 219 485 Β R ³ is C 1 -C 4 alkoxy, hydroxy or hydrogen, R ⁴ is hydrogen, nitro, amino or isothiocyanate, provided that R ² and R ^{4 are} not simultaneously hydrogen but one of R ² and R ⁴ must be hydrogen - and to form complexes containing one of the following metal ions: , Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y or Se, characterized in that said compound of formula IA or a pharmaceutically acceptable salt thereof is complexed with one of said metal ions. 29. The process of claim 28 for the preparation of complexes comprising Sm, Lu or Y as metal ions, wherein the appropriate starting materials are used. 30. The method of claim 28, complexes thereof, those comprising a metal ion: ^L53 Sm>'HO, ⁹ θΥ, of ⁴⁹ Pm, i ⁵⁹ Gd> ⁴ ° La, ¹⁷⁷ Lu, ¹⁷⁵ Yb, ⁴⁷ SC or ¹⁴² Pr, characterized in that the appropriate starting materials are used. 31. The process of claim 28 for the preparation of complexes comprising the following metal ions: ¹⁵³ Sm, ¹⁷⁷ Lu or ⁹⁰ Y, characterized in that the appropriate starting materials are used. 32. A process for the preparation of complexes according to claim 30 or 31 which comprises as a ligand a compound of formula IA prepared according to any one of claims 7, 10, 12, 13 or 26, characterized in that starting materials are used. 33. The process according to claim 28 for the preparation of complexes in which the metal ion is complexed with the ligand prepared according to claim 20, characterized in that the appropriate starting materials are used. 34. A process for the preparation of complexes according to claim 28, wherein the metal ^{ions are} : ^1S3 Sm or Y, characterized in that the appropriate starting materials are used. The process for preparing complexes according to claim 28, wherein the metal ion is complexed with the ligand prepared according to claim 21, wherein the appropriate starting materials are used. 36. The process of claim 35 for the preparation of complexes containing ¹⁵³ Sm per metal ion, wherein the appropriate starting materials are used. The process for preparing complexes according to claim 28, wherein the metal ion is complexed with a ligand prepared according to claim 24 or 25, characterized in that the appropriate starting materials are used. 38. The process of claim 28 for the preparation of complexes comprising ¹⁵³ Sm, ⁹⁰ Y or ¹⁷⁷ Lu per metal ion, wherein the appropriate starting materials are used. 39. The process of claim 28 for the preparation of complexes in which the metal ion is complexed with the ligand prepared according to claim 24, wherein the appropriate starting materials are used. 40. The process of claim 39 for the preparation of complexes comprising ¹⁵³ Sm, ⁹⁰ Y or ¹⁷⁷ Lu per metal ion, wherein the appropriate starting materials are used. 41. A process for the preparation of complexes according to claim 28, wherein the metal ion is complexed with the ligand prepared according to claim 26, wherein the appropriate starting materials are used. 42. The process of claim 41, wherein the metal ion is ¹⁵³ Sm, wherein the appropriate starting materials are used. 43. The process of claim 28 for the preparation of complexes in which the metal ion is complexed with the ligand prepared by the process of claim 27, wherein the appropriate starting materials are used. 44. A process for the preparation of a complex according to claim 43, wherein the metal ion is ¹⁵³ Sm, wherein the appropriate starting materials are used. 45. The process of claim 28 for the preparation of complexes in which the metal ion is complexed with the ligand prepared by the process of claim 15, wherein the appropriate starting materials are used. 46. A process for the preparation of complexes according to claim 45, wherein the metal ion is ¹⁵³ Sm, Y or ¹⁷⁷ Lu, wherein the appropriate starting materials are used. 47. The process of claim 28 for the preparation of complexes in which the metal ion is complexed with the ligand prepared by the process of claim 16, wherein the appropriate starting materials are used. 48. A process for the preparation of a compound of formula IA as a ligand Q is independently hydrogen or (CHR ⁵ ) _p CO ₂ R, Q ¹ is hydrogen or -CO ₂ R, in which R is hydrogen or (C 1 -C 4) -alkyl, provided that Q and Q ¹ together are not at least two hydrogen, and when Q * is hydrogen, R ³ must be other than hydrogen, R ⁵ is hydrogen or C 1 -C 4 alkyl, n is a number from 0 to 5, p is 1 or 2, R ² is hydrogen, amino or isothiocyanate, R ³ is C 1-4 alkoxy, hydroxy or hydrogen, HU 219 485 Β R ⁴ is hydrogen, amino or isothiocyanate, provided that R ² and R ^{4 are} not simultaneously hydrogen but one of R ² and R ⁴ must be hydrogen - one of the following metal ions: La, Ce, Pr, Nd , Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y or Se and conjugates containing an antibody or antibody fragment, characterized in that: a) coupling a compound of formula IA or a pharmaceutically acceptable salt thereof with a metal ion in complexed form to a antibody or antibody fragment thereof, or b) covalently conjugating a compound of formula IA or a pharmaceutically acceptable salt thereof with an antibody or antibody fragment and complexing with a metal ion as above. 49. the preparation method according to claim 48 conjugates containing the following metal ion is ¹⁵³ Sm, ^L66 Ho, Y, ¹⁴ 'Pm'«Gd> ⁴ <> La, ^L77 Lu, ^L75 Yb, ⁴⁷ Sc or ¹⁴² Pr, characterized in that the appropriate starting materials are used. 50. A process for the preparation of conjugates according to claim 49, comprising ¹⁵³ Sm, ⁹⁰ Y or ¹⁷⁷ Lu per metal ion, wherein the appropriate starting materials are used. 51. A 48-50. A process for the preparation of conjugates according to any one of claims 1 to 5, comprising the monoclonal antibody or fragment thereof as an antibody or antibody fragment, characterized in that the appropriate starting materials are used. 52. The method of claim 51 for the preparation of conjugates comprising, as an antibody or antibody fragment, CC-49 (ATCC HB 9459), CC-49 F (ab ') ₂ , CC-83 (ATCC HB 9453), CC- 83 F (ab ') ₂ or B72.3 (ATCC HB 8108), wherein the appropriate starting materials are used. 53. A 48-50. A process for the preparation of conjugates according to any one of claims 1 to 6, comprising as ligand the ligand prepared according to any one of claims 7, 10, 12, 13 or 26, wherein the appropriate starting materials are used. 54. A 49-52. A process for the preparation of conjugates according to any one of claims 1 to 3, comprising the ligand prepared according to claim 19 complexed with a metal ion of ¹⁵³ Sm, characterized in that the appropriate starting materials are used. 55. A 49-52. A process for the preparation of conjugates according to claim 1, wherein the compound prepared according to claim 24 is complexed with one of the following metal ions: ¹⁵³ Sm, Y or ¹⁷⁷ Lu, characterized in that the appropriate starting materials are used. 56. A 49-52. A process for the preparation of a conjugate according to any one of claims 1 to 5, wherein the ligand prepared according to claim 27 is complexed with a metal ion ¹⁵³ Sm, characterized in that the appropriate starting materials are used. 57. A 49-52. A process for the preparation of conjugates according to any one of claims 1 to 4, wherein the compound prepared according to claim 14 is complexed with a metal ion of ¹⁵³ Sm, Y or ¹⁷⁷ Lu, characterized in that the appropriate starting materials are used. 58. A process for the preparation of a pharmaceutical composition comprising the steps of 28-47. A complex prepared according to any one of claims 1 to 6, admixed with a pharmaceutically acceptable carrier and converted to a pharmaceutical composition for administration. 59. A process for the preparation of a pharmaceutical composition comprising the steps of 48-57. The conjugate produced by the process of any one of claims 1 to 6 is mixed with a pharmaceutically acceptable carrier and converted into a pharmaceutical composition for administration. 60. A diagnostic composition, wherein the diagnostic composition of any one of claims 28-47. A complex prepared according to any one of claims 1 to 6 in admixture with a pharmaceutically acceptable carrier. 61. A diagnostic composition, wherein the diagnostic composition of any one of claims 48-57. Conjugate prepared according to any one of claims 1 to 6 in admixture with a pharmaceutically acceptable carrier.